Novel cell contact between podocyte microprojections and parietal epithelial cells analyzed by volume electron microscopy

2020 ◽  
Vol 318 (5) ◽  
pp. F1246-F1251
Author(s):  
Christoph Wrede ◽  
Jan Hegermann ◽  
Christian Mühlfeld
Keyword(s):  
Epithelial Cells ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Cell Contact ◽  
Apical Surface ◽  
Electron Microscopic ◽  
Vascular Perfusion ◽  
Scanning Em ◽  
Parietal Epithelial Cells ◽  
Urinary Space

Podocytes are highly specialized cells with a clear cell polarity. It is known that in health and disease, microvilli protrude from the apical surface of the podocytes into the urinary space. As a basis to better understand the podocyte microprojections/microvilli, the present study analyzed their spatial localization, extension, and contact site with parietal epithelial cells (PECs). Using different electron microscopic (EM) techniques, we analyzed renal corpuscles of healthy young adult male C57BL/6 mice fixed by vascular perfusion. Serial block-face scanning EM was used to visualize entire corpuscles, focused ion beam scanning EM was performed to characterize microprojection/microvilli-rich regions at higher magnification, and transmission EM of serial sections was used to analyze the contact zone between podocyte microprojections and PECs. Numerous microprojections originating from the primary processes of podocytes were present in the urinary space in all regions of the corpuscle. They often reached the apical surface of the PEC but did not make junctional contacts. At high resolution, it was observed that the glycocalyx of both cells was in contact. Depending on the distance between podocytes and PECs, these microprojections had a stretched or coiled state. The present study shows that microprojections/microvilli of podocytes are a physiological feature of healthy mouse kidneys and are frequently in contact with the apical surface of PECs, thus spanning the urinary space. It is proposed that podocyte microprojections serve mechanosensory or communicative functions between podocytes and PECs.

scholarly journals High-precision targeting workflow for volume electron microscopy

2021 ◽  
Vol 220 (9) ◽  
Author(s):  
Paolo Ronchi ◽  
Giulia Mizzon ◽  
Pedro Machado ◽  
Edoardo D’Imprima ◽  
Benedikt T. Best ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Single Cells ◽  
Ion Beam ◽  
Mouse Mammary Gland ◽  
Fluorescence Signal ◽  
3D Objects ◽  
Scanning Em ◽  
Automated Data Acquisition ◽  
Volume Electron Microscopy ◽  
Volume Em

Cells are 3D objects. Therefore, volume EM (vEM) is often crucial for correct interpretation of ultrastructural data. Today, scanning EM (SEM) methods such as focused ion beam (FIB)–SEM are frequently used for vEM analyses. While they allow automated data acquisition, precise targeting of volumes of interest within a large sample remains challenging. Here, we provide a workflow to target FIB-SEM acquisition of fluorescently labeled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeted trimming guided by confocal maps of the fluorescence signal in the resin block. Laser branding is used to create landmarks on the block surface to position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as 3D cultures of mouse mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae, and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before.

scholarly journals Trafficking of Shigella Lipopolysaccharide in Polarized Intestinal Epithelial Cells

1999 ◽  
Vol 145 (4) ◽  
pp. 689-698 ◽  
Author(s):  
Wandy L. Beatty ◽  
Stéphane Méresse ◽  
Pierre Gounon ◽  
Jean Davoust ◽  
Joëlle Mounier ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junctions ◽  
Apical Membrane ◽  
Intestinal Epithelial Cells ◽  
Fluid Phase ◽  
Apical Surface ◽  
Intestinal Epithelial ◽  
Electron Microscopic ◽  
Basolateral Side ◽  
Endocytic Compartments

Bacterial lipopolysaccharide (LPS) at the apical surface of polarized intestinal epithelial cells was previously shown to be transported from the apical to the basolateral pole of the epithelium (Beatty, W.L., and P.J. Sansonetti. 1997. Infect. Immun. 65:4395–4404). The present study was designed to elucidate the transcytotic pathway of LPS and to characterize the endocytic compartments involved in this process. Confocal and electron microscopic analyses revealed that LPS internalized at the apical surface became rapidly distributed within endosomal compartments accessible to basolaterally internalized transferrin. This compartment largely excluded fluid-phase markers added at either pole. Access to the basolateral side of the epithelium subsequent to trafficking to basolateral endosomes occurred via exocytosis into the paracellular space beneath the intercellular tight junctions. LPS appeared to exploit other endocytic routes with much of the internalized LPS recycled to the original apical membrane. In addition, analysis of LPS in association with markers of the endocytic network revealed that some LPS was sent to late endosomal and lysosomal compartments.

scholarly journals HIV corrupts the Cdc42-IQGAP1 axis of actin regulation to promote cell-cell viral spread

2019 ◽  
Author(s):  
Anupriya Aggarwal ◽  
Alberto Ospina Stella ◽  
Catherine Henry ◽  
Kedar Narayan ◽  
Stuart G. Turville
Keyword(s):  
Focused Ion Beam ◽  
Positive Curvature ◽  
Ion Beam ◽  
Membrane Curvature ◽  
Cell Contact ◽  
Cell Contacts ◽  
Viral Spread ◽  
Viral Release ◽  
Viral Mutagenesis ◽  
Cell Cell

AbstractF-Actin remodelling is important for the spread of HIV via cell-cell contacts, yet the mechanisms by which HIV corrupts the actin cytoskeleton are poorly understood. Through live cell imaging and focused ion beam scanning electron microscopy (FIB-SEM), we observed F-Actin structures that exhibit strong positive curvature to be enriched for HIV buds. Virion proteomics, gene silencing, and viral mutagenesis supported a Cdc42-IQGAP1-Arp2/3 pathway as the primary intersection of HIV budding, membrane curvature and F-Actin regulation. Whilst HIV egress activated the Cdc42-Arp2/3 filopodial pathway, this came at the expense of cell-free viral release. Importantly, release could be rescued by cell-cell contact, providing Cdc42 and IQGAP1 were present. From these observations we conclude that out-going HIV has corrupted a central F-Actin node that enables initial coupling of HIV buds to cortical F-Actin to place HIV at the leading cell edge. Whilst this initially prevents particle release, maturation of cell-cell contacts signals back to this F-Actin node to enable viral release & subsequent infection of the contacting cell.

scholarly journals Fusion of Mitochondria to 3-D Networks, Autophagy and Increased Organelle Contacts are Important Subcellular Hallmarks during Cold Stress in Plants

2020 ◽  
Vol 21 (22) ◽  
pp. 8753
Author(s):  
Philip Steiner ◽  
Othmar Buchner ◽  
Ancuela Andosch ◽  
Gerhard Wanner ◽  
Gilbert Neuner ◽  
...  
Keyword(s):  
Low Temperature ◽  
Temperature Stress ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Low Temperature Stress ◽  
Freezing Stress ◽  
Electron Microscopic ◽  
Natural Habitats ◽  
Ultrastructural Alterations ◽  
Micrasterias Denticulata

Low temperature stress has a severe impact on the distribution, physiology, and survival of plants in their natural habitats. While numerous studies have focused on the physiological and molecular adjustments to low temperatures, this study provides evidence that cold induced physiological responses coincide with distinct ultrastructural alterations. Three plants from different evolutionary levels and habitats were investigated: The freshwater alga Micrasterias denticulata, the aquatic plant Lemna sp., and the nival plant Ranunculus glacialis. Ultrastructural alterations during low temperature stress were determined by the employment of 2-D transmission electron microscopy and 3-D reconstructions from focused ion beam–scanning electron microscopic series. With decreasing temperatures, increasing numbers of organelle contacts and particularly the fusion of mitochondria to 3-dimensional networks were observed. We assume that the increase or at least maintenance of respiration during low temperature stress is likely to be based on these mitochondrial interconnections. Moreover, it is shown that autophagy and degeneration processes accompany freezing stress in Lemna and R. glacialis. This might be an essential mechanism to recycle damaged cytoplasmic constituents to maintain the cellular metabolism during freezing stress.

The Fly Brain Atlas

2019 ◽  
Vol 35 (1) ◽  
pp. 637-653 ◽  
Author(s):  
Louis K. Scheffer ◽  
Ian A. Meinertzhagen
Keyword(s):  
Focused Ion Beam ◽  
Optic Lobe ◽  
Ion Beam ◽  
Brain Regions ◽  
Brain Atlas ◽  
Biological Behavior ◽  
Detailed Knowledge ◽  
Motion Sensing ◽  
Electron Microscopic ◽  
Synaptic Circuits

The brain's synaptic networks endow an animal with powerfully adaptive biological behavior. Maps of such synaptic circuits densely reconstructed in those model brains that can be examined and manipulated by genetic means offer the best prospect for understanding the underlying biological bases of behavior. That prospect is now technologically feasible and a scientifically enabling possibility in neurobiology, much as genomics has been in molecular biology and genetics. In Drosophila, two major advances are in electron microscopic technology, using focused ion beam–scanning electron microscopy (FIB-SEM) milling to capture and align digital images, and in computer-aided reconstruction of neuron morphologies. The last decade has witnessed enormous progress in detailed knowledge of the actual synaptic circuits formed by real neurons. Advances in various brain regions that heralded identification of the motion-sensing circuits in the optic lobe are now extending to other brain regions, with the prospect of encompassing the fly's entire nervous system, both brain and ventral nerve cord.

Advanced Physical Analysis Methodology for Yield and Reliability of 28-nm, Bulk-Si, Flip-Chip ICs Using SEM and Backside Deprocessing

2012 ◽  
Author(s):  
Yuanjing (Jane) Li ◽  
Steven Scott ◽  
Howard Lee Marks
Keyword(s):  
Flip Chip ◽  
Focused Ion Beam ◽  
Process Development ◽  
Ion Beam ◽  
Physical Analysis ◽  
Electron Microscopic ◽  
Analysis Methodology ◽  
Processing Techniques ◽  
20 Nm ◽  
Insight Into

Abstract This paper presents a backside chip-level physical analysis methodology using backside de-processing techniques in combination with optimized Scanning Electron Microscopic (SEM) imaging technique and Focused Ion Beam (FIB) cross sectioning to locate and analyze defects and faults in failing IC devices. The case studies illustrate the applications of the method for 28nm flip chip bulk Si CMOS devices and demonstrate how it is used in providing insight into the fab process and design for process and yield improvements. The methods are expected to play an even more important role during 20-nm process development and yield-ramping.

scholarly journals In Situ Structure of Intestinal Apical Surface Reveals Nanovilli on Microvilli

2021 ◽  
Author(s):  
Hao Zhu ◽  
Meijing Li ◽  
Ruixue Zhao ◽  
Ming Li ◽  
Zhaodi Jiang ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Apical Surface ◽  
Supported Membrane ◽  
C Elegans ◽  
Family Protein ◽  
Membrane Protrusions ◽  
Animal Growth

Abstract Microvilli are actin bundle-supported membrane protrusions essential for absorption, secretion, and sensation. Microvilli defects cause human diseases, including gastrointestinal disorders and inherited deafness; however, mechanisms controlling microvilli formation and organization remain unclear. Here, we study microvilli by vitrifying the C. elegans larvae and mouse intestinal tissues with high-pressure freezing, thinning them by cryo-focused ion beam milling, cryo-electron tomography, and sub-tomogram averaging. We uncover that hundreds of previously unrecognized stick-like structures, which we refer to as nanovilli, decorate the lateral surface of C. elegans and mouse microvilli. The C. elegans 37.5-nm long and 4.5-nm wide nanovilli are composed of the protocadherin family protein CDH-8. Loss of nanovilli slows down animal growth and increases the number of Y-shaped microvilli, intermediate structures when a microvillus splits from its tip and separates into two. Our results show that nanovilli space microvilli and suggest a microvilli division model through which microvilli assemble with striking uniformity.

Advanced electron microscopic techniques provide a deeper insight into the peculiar features of podocytes

2015 ◽  
Vol 309 (12) ◽  
pp. F1082-F1089 ◽  
Author(s):  
Tillmann Burghardt ◽  
Florian Hochapfel ◽  
Benjamin Salecker ◽  
Christine Meese ◽  
Hermann-Josef Gröne ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Electron Microscopic ◽  
Glomerular Filtration Barrier ◽  
Filtration Barrier ◽  
Future Work ◽  
Human And Mouse ◽  
Insight Into ◽  
Microscopic Techniques

Podocytes constitute the outer layer of the glomerular filtration barrier, where they form an intricate network of interdigitating foot processes which are connected by slit diaphragms. A hitherto unanswered puzzle concerns the question of whether slit diaphragms are established between foot processes of the same podocyte or between foot processes of different podocytes. By employing focused ion beam-scanning electron microscopy (FIB-SEM), we provide unequivocal evidence that slit diaphragms are formed between foot processes of different podocytes. We extended our investigations of the filtration slit by using dual-axis electron tomography of human and mouse podocytes as well as of Drosophila melanogaster nephrocytes. Using this technique, we not only find a single slit diaphragm which spans the filtration slit around the whole periphery of the foot processes but additional punctate filamentous contacts between adjacent foot processes. Future work will be necessary to determine the proteins constituting the two types of cell-cell contacts.

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