47 results on '"Shtengel G"'
Search Results
2. Examining Mitochondrial Nucleoid Structure and Organization by Correlative Super-Resolution Fluorescence and Electron Microscopy
- Author
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Kopek, B.G., primary, Shtengel, G., additional, Xu, C., additional, Brown, T., additional, Tkachuk, A., additional, Clayton, D., additional, and Hess, H., additional
- Published
- 2012
- Full Text
- View/download PDF
3. Multi-Color Interferometric Photo-Activation Localization Microscopy with Extended Axial Range
- Author
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Shtengel, G, primary, Galbraith, J, additional, Galbraith, C, additional, Kanchanawong, P, additional, Waterman, C, additional, and Hess, H, additional
- Published
- 2011
- Full Text
- View/download PDF
4. Statistical correlation between first and second-order PMD
- Author
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Ibragimov, E., primary, Shtengel, G., additional, and Suh, S., additional
- Published
- 2002
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- View/download PDF
5. Statistical dependence between first and second-order PMD
- Author
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Shtengel, G., primary, Ibragimov, E., additional, Rivera, M., additional, and Suh, S., additional
- Published
- 2001
- Full Text
- View/download PDF
6. High speed packaged electroabsorption modulators for optical communications.
- Author
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Bond, A.E., Shtengel, G., Singh, P., Akulova, Y., and Reynolds, C.L., Jr.
- Published
- 2000
- Full Text
- View/download PDF
7. Differential gain in 1.3-/spl mu/m InGaAsP/InP MQW lasers with p-doped active region
- Author
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Shterengas, L., primary, Reynolds, C.L., additional, Belenky, G., additional, Hybertsen, M., additional, Donetsky, D., additional, and Shtengel, G., additional
- Published
- 2000
- Full Text
- View/download PDF
8. ADVANCES IN MEASUREMENTS OF PHYSICAL PARAMETERS OF SEMICONDUCTOR LASERS
- Author
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SHTENGEL, G. E., primary, KAZARINOV, R. F., additional, BELENKY, G. L., additional, HYBERTSEN, M. S., additional, and ACKERMAN, D. A., additional
- Published
- 1998
- Full Text
- View/download PDF
9. 1.3 µm InGaAsP/InP MQW Lasers for High Temperature Operation Experiment and Modeling
- Author
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Belenky, G., primary, Donetsky, D., additional, Reynolds, C., additional, Shtengel, G., additional, Kazarinov, R., additional, and Luryi, S., additional
- Published
- 1998
- Full Text
- View/download PDF
10. High-speed, low chirp, directly modulated 1.55-/spl mu/m DFB laser sources for 10 Gbit/s local distribution
- Author
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Morton, P.A., primary, Tanbun-Ek, T., additional, Logan, R.A., additional, Ackerman, D.A., additional, Shtengel, G., additional, Chand, N., additional, Johnson, J.E., additional, Yadvish, R.D., additional, Sergent, M., additional, and Sciortino, P.F., additional
- Published
- 1996
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11. Impedance‐corrected carrier lifetime measurements in semiconductor lasers
- Author
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Shtengel, G. E., primary, Ackerman, D. A., additional, Morton, P. A., additional, Flynn, E. J., additional, and Hybertsen, M. S., additional
- Published
- 1995
- Full Text
- View/download PDF
12. Analysis of T0 in 1.3 μm multi‐quantum‐well and bulk active lasers
- Author
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Ackerman, D. A., primary, Morton, P. A., additional, Shtengel, G. E., additional, Hybertsen, M. S., additional, Kazarinov, R. F., additional, Tanbun‐Ek, T., additional, and Logan, R. A., additional
- Published
- 1995
- Full Text
- View/download PDF
13. Threshold carrier density in vertical cavity surface emitting lasers
- Author
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Bae, J. W., primary, Shtengel, G., additional, Kuksenkov, D., additional, Temkin, H., additional, and Brusenbach, P., additional
- Published
- 1995
- Full Text
- View/download PDF
14. Spontaneous emission factor and its scaling in vertical cavity surface emitting lasers
- Author
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Shtengel, G., primary, Temkin, H., additional, Uchida, T., additional, Kim, M., additional, Brusenbach, P., additional, and Parsons, C., additional
- Published
- 1994
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15. High-speed vertical-cavity surface emitting laser
- Author
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Shtengel, G., primary, Temkin, H., additional, Brusenbach, P., additional, Uchida, T., additional, Kim, M., additional, Parsons, C., additional, Quinn, W.E., additional, and Swirhun, S.E., additional
- Published
- 1993
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16. Vertical-cavity surface-emitting lasers with 14 GHz bandwidth
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Brusenbach, P., primary, Uchida, T.K., additional, Parsons, C., additional, Kim, M., additional, Quinn, W.E., additional, Swirhun, S.E., additional, Shtengel, G., additional, and Temkin, H., additional
- Published
- 1993
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17. Interferometric fluorescent super-resolution microscopy resolves 3D cellular nano-architecture
- Author
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Shtengel, G., Galbraith, J. A., Galbraith, C. G., Lippincott-Schwartz, J., Gillette, J. M., Manley, Suliana, Sougrat, R., Waterman, C. M., Kanchanawong, P., Davidson, M. W., Fetter, R. D., and Hess, H. F.
- Subjects
BIO-IMAGING
18. Statistical dependence between first and second-order PMD
- Author
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Shtengel, G., primary, Ibragimov, E., additional, Rivera, M., additional, and Suh, S., additional
- Full Text
- View/download PDF
19. 1.3μm InGaAsP/InP MQW Lasers for High Temperature Operation. Experiment and Modeling
- Author
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Belenky, G., primary, Donetsky, D., additional, Reynolds, C., additional, Shtengel, G., additional, Kazarinov, R., additional, and Luryi, S., additional
- Full Text
- View/download PDF
20. High speed packaged electroabsorption modulators for optical communications
- Author
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Bond, A.E., primary, Shtengel, G., additional, Singh, P., additional, Akulova, Y., additional, and Reynolds, C.L., additional
- Full Text
- View/download PDF
21. Differential gain in 1.3-μm InGaAsP/InP MQW lasers with p-doped active region.
- Author
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Shterengas, L., Reynolds, C.L., Jr., Belenky, G., Hybertsen, M., Donetsky, D., and Shtengel, G.
- Published
- 2000
- Full Text
- View/download PDF
22. 1.3/spl mu/m InGaAsP/InP MQW Lasers for High Temperature Operation. Experiment and Modeling.
- Author
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Belenky, G., Donetsky, D., Reynolds, C., Shtengel, G., Kazarinov, R., and Luryi, S.
- Published
- 1998
- Full Text
- View/download PDF
23. TE and TM optical gains in AlGaAs/GaAs single-quantum-well lasers.
- Author
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Avrutin, E. A., Chebunina, I. E., Eliachevitch, I. A., Gurevich, S. A., Portnoi, M. E., and Shtengel, G. E.
- Published
- 1993
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- View/download PDF
24. Transport and Organization of Individual Vimentin Filaments Within Dense Networks Revealed by Single Particle Tracking and 3D FIB-SEM.
- Author
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Renganathan B, Moore A, Yeo WH, Petruncio A, Ackerman D, Wiegel A, Pasolli HA, Xu CS, Shtengel G, Hess HF, Serpinskaya AS, Zhang HF, Lippincott-Schwartz J, and Gelfand VI
- Abstract
Single-particle tracking demonstrates that individual filaments in bundles of vimentin intermediate filaments are transported in the cytoplasm by motor proteins along microtubules. Furthermore, using 3D FIB-SEM the authors showed that vimentin filament bundles are loosely packed and coaligned with microtubules. 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 μm
3 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., Competing Interests: Statements and Declarations All authors declare no conflict of interest.- Published
- 2024
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25. COPII with ALG2 and ESCRTs control lysosome-dependent microautophagy of ER exit sites.
- Author
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Liao YC, Pang S, Li WP, Shtengel G, Choi H, Schaefer K, Xu CS, and Lippincott-Schwartz J
- Subjects
- Humans, Cell Cycle Proteins metabolism, Cell Cycle Proteins genetics, Protein Transport, HeLa Cells, Apoptosis Regulatory Proteins metabolism, Apoptosis Regulatory Proteins genetics, Autophagy physiology, TOR Serine-Threonine Kinases metabolism, Calcium metabolism, Lysosomes metabolism, Endoplasmic Reticulum metabolism, Endosomal Sorting Complexes Required for Transport metabolism, Endosomal Sorting Complexes Required for Transport genetics, Calcium-Binding Proteins metabolism, Calcium-Binding Proteins genetics, COP-Coated Vesicles metabolism, Microautophagy, Vesicular Transport Proteins metabolism, Vesicular Transport Proteins genetics
- Abstract
Endoplasmic reticulum exit sites (ERESs) are tubular outgrowths of endoplasmic reticulum that serve as the earliest station for protein sorting and export into the secretory pathway. How these structures respond to different cellular conditions remains unclear. Here, we report that ERESs undergo lysosome-dependent microautophagy when Ca
2+ is released by lysosomes in response to nutrient stressors such as mTOR inhibition or amino acid starvation in mammalian cells. Targeting and uptake of ERESs into lysosomes were observed by super-resolution live-cell imaging and focus ion beam scanning electron microscopy (FIB-SEM). The mechanism was ESCRT dependent and required ubiquitinated SEC31, ALG2, and ALIX, with a knockout of ALG2 or function-blocking mutations of ALIX preventing engulfment of ERESs by lysosomes. In vitro, reconstitution of the pathway was possible using lysosomal lipid-mimicking giant unilamellar vesicles and purified recombinant components. Together, these findings demonstrate a pathway of lysosome-dependent ERES microautophagy mediated by COPII, ALG2, and ESCRTS induced by nutrient stress., Competing Interests: Declaration of interests Portions of the technology described here are covered by US Patent 10,600,615 titled “Enhanced FIB-SEM systems for large-volume 3D imaging,” which was issued to C.S.X., K.J.H., and H.F.H. and assigned to Howard Hughes Medical Institute on 24 March 2020., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2024
- Full Text
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26. Motion of VAPB molecules reveals ER-mitochondria contact site subdomains.
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Obara CJ, Nixon-Abell J, Moore AS, Riccio F, Hoffman DP, Shtengel G, Xu CS, Schaefer K, Pasolli HA, Masson JB, Hess HF, Calderon CP, Blackstone C, and Lippincott-Schwartz J
- Subjects
- Humans, Amyotrophic Lateral Sclerosis genetics, Signal Transduction, Microscopy, Electron, Imaging, Three-Dimensional, Binding Sites, Diffusion, Time Factors, Mutation, Homeostasis, Endoplasmic Reticulum chemistry, Endoplasmic Reticulum metabolism, Endoplasmic Reticulum ultrastructure, Mitochondria chemistry, Mitochondria metabolism, Mitochondria ultrastructure, Mitochondrial Membranes chemistry, Mitochondrial Membranes metabolism, Mitochondrial Membranes ultrastructure, Vesicular Transport Proteins genetics, Vesicular Transport Proteins metabolism, Vesicular Transport Proteins ultrastructure, Movement
- Abstract
To coordinate cellular physiology, eukaryotic cells rely on the rapid exchange of molecules at specialized organelle-organelle contact sites
1,2 . Endoplasmic reticulum-mitochondrial contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signalling molecules, lipids and metabolites3,4 . ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle5,6 . However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation7,8 , 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., (© 2024. The Author(s).)- Published
- 2024
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27. ESCRT-mediated membrane repair protects tumor-derived cells against T cell attack.
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Ritter AT, Shtengel G, Xu CS, Weigel A, Hoffman DP, Freeman M, Iyer N, Alivodej N, Ackerman D, Voskoboinik I, Trapani J, Hess HF, and Mellman I
- Subjects
- Granzymes metabolism, Perforin genetics, Perforin metabolism, Pore Forming Cytotoxic Proteins genetics, Pore Forming Cytotoxic Proteins metabolism, T-Lymphocytes, Cytotoxic metabolism, Endosomal Sorting Complexes Required for Transport genetics, Endosomal Sorting Complexes Required for Transport metabolism, Membrane Glycoproteins metabolism
- Abstract
Cytotoxic T lymphocytes (CTLs) and natural killer cells kill virus-infected and tumor cells through the polarized release of perforin and granzymes. Perforin is a pore-forming toxin that creates a lesion in the plasma membrane of the target cell through which granzymes enter the cytosol and initiate apoptosis. Endosomal sorting complexes required for transport (ESCRT) proteins are involved in the repair of small membrane wounds. We found that ESCRT proteins were precisely recruited in target cells to sites of CTL engagement immediately after perforin release. Inhibition of ESCRT machinery in cancer-derived cells enhanced their susceptibility to CTL-mediated killing. Thus, repair of perforin pores by ESCRT machinery limits granzyme entry into the cytosol, potentially enabling target cells to resist cytolytic attack.
- Published
- 2022
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28. Publisher Correction: An open-access volume electron microscopy atlas of whole cells and tissues.
- Author
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Xu CS, Pang S, Shtengel G, Müller A, Ritter AT, Hoffman HK, Takemura SY, Lu Z, Pasolli HA, Iyer N, Chung J, Bennett D, Weigel AV, Freeman M, van Engelenburg SB, Walther TC, Farese RV Jr, Lippincott-Schwartz J, Mellman I, Solimena M, and Hess HF
- Published
- 2021
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29. An open-access volume electron microscopy atlas of whole cells and tissues.
- Author
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Xu CS, Pang S, Shtengel G, Müller A, Ritter AT, Hoffman HK, Takemura SY, Lu Z, Pasolli HA, Iyer N, Chung J, Bennett D, Weigel AV, Freeman M, van Engelenburg SB, Walther TC, Farese RV Jr, Lippincott-Schwartz J, Mellman I, Solimena M, and Hess HF
- Subjects
- Animals, Cell Line, Cells, Cultured, Drosophila melanogaster cytology, Drosophila melanogaster ultrastructure, Female, Golgi Apparatus ultrastructure, Humans, Interphase, Islets of Langerhans cytology, Male, Mice, Microtubules ultrastructure, Neuroglia ultrastructure, Neurons ultrastructure, Open Access Publishing, Ovarian Neoplasms immunology, Ovarian Neoplasms ultrastructure, Ribosomes ultrastructure, Synaptic Vesicles ultrastructure, T-Lymphocytes, Cytotoxic cytology, T-Lymphocytes, Cytotoxic immunology, T-Lymphocytes, Cytotoxic ultrastructure, Datasets as Topic, Information Dissemination, Microscopy, Electron, Scanning methods, Microscopy, Electron, Scanning standards, Organelles ultrastructure
- Abstract
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
1 . 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 OpenOrganelle2 ) 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., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)- Published
- 2021
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30. ER-to-Golgi protein delivery through an interwoven, tubular network extending from ER.
- Author
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Weigel AV, Chang CL, Shtengel G, Xu CS, Hoffman DP, Freeman M, Iyer N, Aaron J, Khuon S, Bogovic J, Qiu W, Hess HF, and Lippincott-Schwartz J
- Subjects
- Biological Transport, Active, HeLa Cells, Humans, Protein Transport, COP-Coated Vesicles metabolism, Endoplasmic Reticulum metabolism, Golgi Apparatus metabolism, Microtubules metabolism, Ubiquitin-Protein Ligases metabolism
- Abstract
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., Competing Interests: Declaration of interests C.S.X. and H.F.H. have a U.S. patent 10,600,615 of the enhanced FIB-SEM system used in this work., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
- Full Text
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31. Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells.
- Author
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Hoffman DP, Shtengel G, Xu CS, Campbell KR, Freeman M, Wang L, Milkie DE, Pasolli HA, Iyer N, Bogovic JA, Stabley DR, Shirinifard A, Pang S, Peale D, Schaefer K, Pomp W, Chang CL, Lippincott-Schwartz J, Kirchhausen T, Solecki DJ, Betzig E, and Hess HF
- Subjects
- Animals, COS Cells, Cell Adhesion, Cell Line, Tumor, Chlorocebus aethiops, Freezing, HeLa Cells, Humans, Mice, Cells ultrastructure, Cryoelectron Microscopy methods, Imaging, Three-Dimensional methods, Microscopy, Fluorescence methods
- Abstract
Within cells, the spatial compartmentalization of thousands of distinct proteins serves a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) can elucidate protein spatial relationships to global ultrastructure, but has suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional cryogenic SR and focused ion beam-milled block-face EM across entire vitreously frozen cells. The approach preserves ultrastructure while enabling independent SR and EM workflow optimization. We discovered unexpected protein-ultrastructure relationships in mammalian cells including intranuclear vesicles containing endoplasmic reticulum-associated proteins, web-like adhesions between cultured neurons, and chromatin domains subclassified on the basis of transcriptional activity. Our findings illustrate the value of a comprehensive multimodal view of ultrastructural variability across whole cells., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)
- Published
- 2020
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32. Spastin tethers lipid droplets to peroxisomes and directs fatty acid trafficking through ESCRT-III.
- Author
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Chang CL, Weigel AV, Ioannou MS, Pasolli HA, Xu CS, Peale DR, Shtengel G, Freeman M, Hess HF, Blackstone C, and Lippincott-Schwartz J
- Subjects
- ATP Binding Cassette Transporter, Subfamily D, Member 1 metabolism, Adenosine Triphosphatases metabolism, Amino Acid Motifs, Biological Transport, HeLa Cells, Humans, Hydrolysis, Lauric Acids metabolism, Models, Biological, Mutant Proteins metabolism, Oncogene Proteins metabolism, Spastin chemistry, Endosomal Sorting Complexes Required for Transport metabolism, Fatty Acids metabolism, Lipid Droplets metabolism, Peroxisomes metabolism, Spastin metabolism
- Abstract
Lipid droplets (LDs) are neutral lipid storage organelles that transfer lipids to various organelles including peroxisomes. Here, we show that the hereditary spastic paraplegia protein M1 Spastin, a membrane-bound AAA ATPase found on LDs, coordinates fatty acid (FA) trafficking from LDs to peroxisomes through two interrelated mechanisms. First, M1 Spastin forms a tethering complex with peroxisomal ABCD1 to promote LD-peroxisome contact formation. Second, M1 Spastin recruits the membrane-shaping ESCRT-III proteins IST1 and CHMP1B to LDs via its MIT domain to facilitate LD-to-peroxisome FA trafficking, possibly through IST1- and CHMP1B-dependent modifications in LD membrane morphology. Furthermore, LD-to-peroxisome FA trafficking mediated by M1 Spastin is required to relieve LDs of lipid peroxidation. M1 Spastin's dual roles in tethering LDs to peroxisomes and in recruiting ESCRT-III components to LD-peroxisome contact sites for FA trafficking may underlie the pathogenesis of diseases associated with defective FA metabolism in LDs and peroxisomes., (© 2019 Chang et al.)
- Published
- 2019
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33. Diverse protocols for correlative super-resolution fluorescence imaging and electron microscopy of chemically fixed samples.
- Author
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Kopek BG, Paez-Segala MG, Shtengel G, Sochacki KA, Sun MG, Wang Y, Xu CS, van Engelenburg SB, Taraska JW, Looger LL, and Hess HF
- Subjects
- Biological Products analysis, Cytological Techniques methods, Microscopy, Electron methods, Optical Imaging methods
- Abstract
Our groups have recently developed related approaches for sample preparation for super-resolution imaging within endogenous cellular environments using correlative light and electron microscopy (CLEM). Four distinct techniques for preparing and acquiring super-resolution CLEM data sets for aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. The choice of the best protocol for a given application depends on a number of criteria that are discussed in detail. Tokuyasu cryosectioning is relatively rapid but is limited to small, delicate specimens. Whole-cell mount has the simplest sample preparation but is restricted to surface structures. Cell unroofing and platinum replication creates high-contrast, 3D images of the cytoplasmic surface of the plasma membrane but is more challenging than whole-cell mount. Resin embedding permits serial sectioning of large samples but is limited to osmium-resistant probes, and is technically difficult. Expected results from these protocols include super-resolution localization (∼10-50 nm) of fluorescent targets within the context of electron microscopy ultrastructure, which can help address cell biological questions. These protocols can be completed in 2-7 d, are compatible with a number of super-resolution imaging protocols, and are broadly applicable across biology.
- Published
- 2017
- Full Text
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34. Synthesis of a Far-Red Photoactivatable Silicon-Containing Rhodamine for Super-Resolution Microscopy.
- Author
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Grimm JB, Klein T, Kopek BG, Shtengel G, Hess HF, Sauer M, and Lavis LD
- Subjects
- Microscopy, Fluorescence methods, Rhodamines chemistry, Silicon analysis
- Abstract
The rhodamine system is a flexible framework for building small-molecule fluorescent probes. Changing N-substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si-containing analogue of Q-rhodamine. This probe is the first example of a "caged" Si-rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red-shifted to allow multicolor imaging. The dye is a useful label for super-resolution imaging and constitutes a new scaffold for far-red fluorogenic molecules., (© 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)
- Published
- 2016
- Full Text
- View/download PDF
35. Molecular mechanism of vinculin activation and nanoscale spatial organization in focal adhesions.
- Author
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Case LB, Baird MA, Shtengel G, Campbell SL, Hess HF, Davidson MW, and Waterman CM
- Subjects
- Actins chemistry, Actins metabolism, Blotting, Western, Cell Line, Cell Line, Tumor, Fluorescence Resonance Energy Transfer, Focal Adhesions genetics, Humans, Integrins chemistry, Integrins metabolism, Luminescent Proteins genetics, Luminescent Proteins metabolism, Microscopy, Fluorescence methods, Models, Molecular, Mutation, Paxillin chemistry, Paxillin genetics, Paxillin metabolism, Protein Binding, Protein Structure, Tertiary, RNA Interference, Talin chemistry, Talin genetics, Talin metabolism, Vinculin chemistry, Vinculin genetics, Focal Adhesions metabolism, Nanostructures, Nanotechnology methods, Vinculin metabolism
- Abstract
Focal adhesions (FAs) link the extracellular matrix to the actin cytoskeleton to mediate cell adhesion, migration, mechanosensing and signalling. FAs have conserved nanoscale protein organization, suggesting that the position of proteins within FAs regulates their activity and function. Vinculin binds different FA proteins to mediate distinct cellular functions, but how vinculin's interactions are spatiotemporally organized within FAs is unknown. Using interferometric photoactivation localization super-resolution microscopy to assay vinculin nanoscale localization and a FRET biosensor to assay vinculin conformation, we found that upward repositioning within the FA during FA maturation facilitates vinculin activation and mechanical reinforcement of FAs. Inactive vinculin localizes to the lower integrin signalling layer in FAs by binding to phospho-paxillin. Talin binding activates vinculin and targets active vinculin higher in FAs where vinculin can engage retrograde actin flow. Thus, specific protein interactions are spatially segregated within FAs at the nanoscale to regulate vinculin activation and function.
- Published
- 2015
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36. A multi-layered protein network stabilizes the Escherichia coli FtsZ-ring and modulates constriction dynamics.
- Author
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Buss J, Coltharp C, Shtengel G, Yang X, Hess H, and Xiao J
- Subjects
- Carrier Proteins metabolism, Cell Cycle Proteins metabolism, Chromosomal Proteins, Non-Histone metabolism, Escherichia coli physiology, Escherichia coli ultrastructure, Escherichia coli Proteins metabolism, Motion, Bacterial Proteins metabolism, Cell Division, Cytoskeletal Proteins metabolism, Escherichia coli metabolism
- Abstract
The prokaryotic tubulin homolog, FtsZ, forms a ring-like structure (FtsZ-ring) at midcell. The FtsZ-ring establishes the division plane and enables the assembly of the macromolecular division machinery (divisome). Although many molecular components of the divisome have been identified and their interactions extensively characterized, the spatial organization of these proteins within the divisome is unclear. Consequently, the physical mechanisms that drive divisome assembly, maintenance, and constriction remain elusive. Here we applied single-molecule based superresolution imaging, combined with genetic and biophysical investigations, to reveal the spatial organization of cellular structures formed by four important divisome proteins in E. coli: FtsZ, ZapA, ZapB and MatP. We show that these interacting proteins are arranged into a multi-layered protein network extending from the cell membrane to the chromosome, each with unique structural and dynamic properties. Further, we find that this protein network stabilizes the FtsZ-ring, and unexpectedly, slows down cell constriction, suggesting a new, unrecognized role for this network in bacterial cell division. Our results provide new insight into the structure and function of the divisome, and highlight the importance of coordinated cell constriction and chromosome segregation.
- Published
- 2015
- Full Text
- View/download PDF
37. Fixation-resistant photoactivatable fluorescent proteins for CLEM.
- Author
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Paez-Segala MG, Sun MG, Shtengel G, Viswanathan S, Baird MA, Macklin JJ, Patel R, Allen JR, Howe ES, Piszczek G, Hess HF, Davidson MW, Wang Y, and Looger LL
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, CHO Cells, Cricetulus, Fluorescence, HeLa Cells, Humans, Luminescent Proteins genetics, Molecular Imaging methods, Molecular Sequence Data, Osmium Tetroxide chemistry, Photochemistry methods, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Luminescent Proteins metabolism, Microscopy, Electron, Scanning methods, Microscopy, Electron, Transmission methods
- Abstract
Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they lose their fluorescence after heavy fixation, hindering applications such as correlative light and electron microscopy (CLEM). Here we report engineered variants of the photoconvertible Eos fluorescent protein that fluoresce and photoconvert normally in heavily fixed (0.5-1% OsO4), plastic resin-embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy.
- Published
- 2015
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38. Anesthetized- and awake-patched whole-cell recordings in freely moving rats using UV-cured collar-based electrode stabilization.
- Author
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Lee D, Shtengel G, Osborne JE, and Lee AK
- Subjects
- Anesthesia methods, Animals, Behavior, Animal, Cytological Techniques instrumentation, Electrodes, Electrophysiology instrumentation, Equipment Design, Hippocampus cytology, Motor Activity, Neurons cytology, Rats, Wistar, Reproducibility of Results, Ultraviolet Rays, Wakefulness, Cytological Techniques methods, Electrophysiology methods, Neurons physiology
- Abstract
Intracellular recording allows precise measurement and manipulation of individual neurons, but it requires stable mechanical contact between the electrode and the cell membrane, and thus it has remained challenging to perform in behaving animals. Whole-cell recordings in freely moving animals can be obtained by rigidly fixing ('anchoring') the pipette electrode to the head; however, previous anchoring procedures were slow and often caused substantial pipette movement, resulting in loss of the recording or of recording quality. We describe a UV-transparent collar and UV-cured adhesive technique that rapidly (within 15 s) anchors pipettes in place with virtually no movement, thus substantially improving the reliability, yield and quality of freely moving whole-cell recordings. Recordings are first obtained from anesthetized or awake head-fixed rats. UV light cures the thin adhesive layers linking pipette to collar to head. Then, the animals are rapidly and smoothly released for recording during unrestrained behavior. The anesthetized-patched version can be completed in ∼4-7 h (excluding histology) and the awake-patched version requires ∼1-4 h per day for ∼2 weeks. These advances should greatly facilitate studies of neuronal integration and plasticity in identified cells during natural behaviors.
- Published
- 2014
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- View/download PDF
39. Correlative super-resolution fluorescence and metal-replica transmission electron microscopy.
- Author
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Sochacki KA, Shtengel G, van Engelenburg SB, Hess HF, and Taraska JW
- Subjects
- Adaptor Proteins, Vesicular Transport metabolism, Adaptor Proteins, Vesicular Transport ultrastructure, Clathrin ultrastructure, Humans, Membrane Proteins metabolism, Gold chemistry, Membrane Proteins ultrastructure, Microscopy, Electron, Transmission, Microscopy, Fluorescence, Nanotubes chemistry
- Abstract
We combine super-resolution localization fluorescence microscopy with transmission electron microscopy of metal replicas to locate proteins on the landscape of the cellular plasma membrane at the nanoscale. We validate robust correlation on the scale of 20 nm by imaging endogenous clathrin (in two and three dimensions) and apply the method to find the previously unknown three-dimensional position of the endocytic protein epsin on clathrin-coated structures at the plasma membrane.
- Published
- 2014
- Full Text
- View/download PDF
40. Distribution of ESCRT machinery at HIV assembly sites reveals virus scaffolding of ESCRT subunits.
- Author
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Van Engelenburg SB, Shtengel G, Sengupta P, Waki K, Jarnik M, Ablan SD, Freed EO, Hess HF, and Lippincott-Schwartz J
- Subjects
- HIV-1 metabolism, Humans, Imaging, Three-Dimensional methods, Microscopy methods, Protein Subunits metabolism, Virion metabolism, Virus Release, gag Gene Products, Human Immunodeficiency Virus metabolism, Endosomal Sorting Complexes Required for Transport metabolism, HIV Infections virology, HIV-1 physiology, Virion physiology, Virus Assembly
- Abstract
The human immunodeficiency virus (HIV) hijacks the endosomal sorting complexes required for transport (ESCRT) to mediate virus release from infected cells. The nanoscale organization of ESCRT machinery necessary for mediating viral abscission is unclear. Here, we applied three-dimensional superresolution microscopy and correlative electron microscopy to delineate the organization of ESCRT components at HIV assembly sites. We observed ESCRT subunits localized within the head of budding virions and released particles, with head-localized levels of CHMP2A decreasing relative to Tsg101 and CHMP4B upon virus abscission. Thus, the driving force for HIV release may derive from initial scaffolding of ESCRT subunits within the viral bud interior followed by plasma membrane association and selective remodeling of ESCRT subunits.
- Published
- 2014
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- View/download PDF
41. Imaging cellular ultrastructure by PALM, iPALM, and correlative iPALM-EM.
- Author
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Shtengel G, Wang Y, Zhang Z, Goh WI, Hess HF, and Kanchanawong P
- Subjects
- Animals, Cell Line, Fiducial Markers, Gold chemistry, Humans, Metal Nanoparticles chemistry, Microscopy, Electron, Microscopy, Fluorescence methods, Fluorescent Dyes chemistry, Single-Cell Analysis methods
- Abstract
Many biomolecules in cells can be visualized with high sensitivity and specificity by fluorescence microscopy. However, the resolution of conventional light microscopy is limited by diffraction to ~200-250 nm laterally and >500 nm axially. Here, we describe superresolution methods based on single-molecule localization analysis of photoswitchable fluorophores (PALM: photoactivated localization microscopy) as well as our recent three-dimensional (3D) method (iPALM: interferometric PALM) that allows imaging with a resolution better than 20 nm in all three dimensions. Considerations for their implementations, applications to multicolor imaging, and a recent development that extend the imaging depth of iPALM to ~750 nm are discussed. As the spatial resolution of superresolution fluorescence microscopy converges with that of electron microscopy (EM), direct imaging of the same specimen using both approaches becomes feasible. This could be particularly useful for cross validation of experiments, and thus, we also describe recent methods that were developed for correlative superresolution fluorescence and EM., (© 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
- Full Text
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42. Correlative photoactivated localization and scanning electron microscopy.
- Author
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Kopek BG, Shtengel G, Grimm JB, Clayton DA, and Hess HF
- Subjects
- Animals, Cryoultramicrotomy, Imaging, Three-Dimensional, Mice, Microscopy, Electron, Scanning methods, Microscopy, Fluorescence methods, Microtomy methods, NIH 3T3 Cells, Phalloidine chemistry, Staining and Labeling methods, Actins ultrastructure, Microscopy, Electron, Scanning instrumentation, Microscopy, Fluorescence instrumentation, Mitochondria ultrastructure, Nuclear Lamina ultrastructure, Peroxisomes ultrastructure
- Abstract
The ability to localize proteins precisely within subcellular space is crucial to understanding the functioning of biological systems. Recently, we described a protocol that correlates a precise map of fluorescent fusion proteins localized using three-dimensional super-resolution optical microscopy with the fine ultrastructural context of three-dimensional electron micrographs. While it achieved the difficult simultaneous objectives of high photoactivated fluorophore preservation and ultrastructure preservation, it required a super-resolution optical and specialized electron microscope that is not available to many researchers. We present here a faster and more practical protocol with the advantage of a simpler two-dimensional optical (Photoactivated Localization Microscopy (PALM)) and scanning electron microscope (SEM) system that retains the often mutually exclusive attributes of fluorophore preservation and ultrastructure preservation. As before, cryosections were prepared using the Tokuyasu protocol, but the staining protocol was modified to be amenable for use in a standard SEM without the need for focused ion beam ablation. We show the versatility of this technique by labeling different cellular compartments and structures including mitochondrial nucleoids, peroxisomes, and the nuclear lamina. We also demonstrate simultaneous two-color PALM imaging with correlated electron micrographs. Lastly, this technique can be used with small-molecule dyes as demonstrated with actin labeling using phalloidin conjugated to a caged dye. By retaining the dense protein labeling expected for super-resolution microscopy combined with ultrastructural preservation, simplifying the tools required for correlative microscopy, and expanding the number of useful labels we expect this method to be accessible and valuable to a wide variety of researchers.
- Published
- 2013
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43. Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes.
- Author
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Kopek BG, Shtengel G, Xu CS, Clayton DA, and Hess HF
- Subjects
- 3T3 Cells, Animals, DNA, Mitochondrial genetics, DNA, Mitochondrial metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Fibroblasts cytology, Fibroblasts metabolism, High Mobility Group Proteins genetics, High Mobility Group Proteins metabolism, Imaging, Three-Dimensional, Luminescent Proteins genetics, Luminescent Proteins metabolism, Mice, Mitochondria genetics, Mitochondria metabolism, Mitochondria ultrastructure, Mitochondrial Membranes metabolism, Mitochondrial Proteins metabolism, Reproducibility of Results, DNA, Mitochondrial ultrastructure, Microscopy, Electron methods, Microscopy, Fluorescence methods, Microscopy, Interference methods, Mitochondrial Membranes ultrastructure, Mitochondrial Proteins ultrastructure
- Abstract
Microscopic images of specific proteins in their cellular context yield important insights into biological processes and cellular architecture. The advent of superresolution optical microscopy techniques provides the possibility to augment EM with nanometer-resolution fluorescence microscopy to access the precise location of proteins in the context of cellular ultrastructure. Unfortunately, efforts to combine superresolution fluorescence and EM have been stymied by the divergent and incompatible sample preparation protocols of the two methods. Here, we describe a protocol that preserves both the delicate photoactivatable fluorescent protein labels essential for superresolution microscopy and the fine ultrastructural context of EM. This preparation enables direct 3D imaging in 500- to 750-nm sections with interferometric photoactivatable localization microscopy followed by scanning EM images generated by focused ion beam ablation. We use this process to "colorize" detailed EM images of the mitochondrion with the position of labeled proteins. The approach presented here has provided a new level of definition of the in vivo nature of organization of mitochondrial nucleoids, and we expect this straightforward method to be applicable to many other biological questions that can be answered by direct imaging.
- Published
- 2012
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44. Imaging the post-fusion release and capture of a vesicle membrane protein.
- Author
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Sochacki KA, Larson BT, Sengupta DC, Daniels MP, Shtengel G, Hess HF, and Taraska JW
- Subjects
- Animals, Cell Membrane physiology, Cell Membrane ultrastructure, Clathrin physiology, Clathrin ultrastructure, Endocytosis physiology, Exocytosis physiology, Membrane Proteins ultrastructure, Microscopy, Electron, Microscopy, Interference methods, PC12 Cells physiology, Rats, Synaptic Vesicles physiology, Synaptic Vesicles ultrastructure, Vesicular Acetylcholine Transport Proteins ultrastructure, Membrane Fusion physiology, Membrane Proteins physiology, Vesicular Acetylcholine Transport Proteins physiology
- Abstract
The molecular mechanism responsible for capturing, sorting and retrieving vesicle membrane proteins following triggered exocytosis is not understood. Here we image the post-fusion release and then capture of a vesicle membrane protein, the vesicular acetylcholine transporter, from single vesicles in living neuroendocrine cells. We combine these measurements with super-resolution interferometric photo-activation localization microscopy and electron microscopy, and modelling to map the nanometer-scale topography and architecture of the structures responsible for the transporter's capture following exocytosis. We show that after exocytosis, the transporter rapidly diffuses into the plasma membrane, but most travels only a short distance before it is locally captured over a dense network of membrane-resident clathrin-coated structures. We propose that the extreme density of these structures acts as a short-range diffusion trap. They quickly sequester diffusing vesicle material and limit its spread across the membrane. This system could provide a means for clathrin-mediated endocytosis to quickly recycle vesicle proteins in highly excitable cells.
- Published
- 2012
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45. Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction.
- Author
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Brown TA, Tkachuk AN, Shtengel G, Kopek BG, Bogenhagen DF, Hess HF, and Clayton DA
- Subjects
- 3T3 Cells, Animals, Mice, Microscopy, Confocal, Plasmids, Sequence Alignment, Sequence Analysis, DNA, DNA, Mitochondrial chemistry, Microscopy, Fluorescence methods, Mitochondria metabolism, Mitochondrial Proteins chemistry
- Abstract
A fundamental objective in molecular biology is to understand how DNA is organized in concert with various proteins, RNA, and biological membranes. Mitochondria maintain and express their own DNA (mtDNA), which is arranged within structures called nucleoids. Their functions, dimensions, composition, and precise locations relative to other mitochondrial structures are poorly defined. Superresolution fluorescence microscopy techniques that exceed the previous limits of imaging within the small and highly compartmentalized mitochondria have been recently developed. We have improved and employed both two- and three-dimensional applications of photoactivated localization microscopy (PALM and iPALM, respectively) to visualize the core dimensions and relative locations of mitochondrial nucleoids at an unprecedented resolution. PALM reveals that nucleoids differ greatly in size and shape. Three-dimensional volumetric analysis indicates that, on average, the mtDNA within ellipsoidal nucleoids is extraordinarily condensed. Two-color PALM shows that the freely diffusible mitochondrial matrix protein is largely excluded from the nucleoid. In contrast, nucleoids are closely associated with the inner membrane and often appear to be wrapped around cristae or crista-like inner membrane invaginations. Determinations revealing high packing density, separation from the matrix, and tight association with the inner membrane underscore the role of mechanisms that regulate access to mtDNA and that remain largely unknown.
- Published
- 2011
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46. Nanoscale architecture of integrin-based cell adhesions.
- Author
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Kanchanawong P, Shtengel G, Pasapera AM, Ramko EB, Davidson MW, Hess HF, and Waterman CM
- Subjects
- Actins metabolism, Animals, Cell Adhesion, Cell Line, Cell Line, Tumor, Cell Membrane metabolism, Cell Membrane ultrastructure, Extracellular Matrix ultrastructure, Humans, Mice, Models, Biological, Extracellular Matrix metabolism, Integrins metabolism
- Abstract
Cell adhesions to the extracellular matrix (ECM) are necessary for morphogenesis, immunity and wound healing. Focal adhesions are multifunctional organelles that mediate cell-ECM adhesion, force transmission, cytoskeletal regulation and signalling. Focal adhesions consist of a complex network of trans-plasma-membrane integrins and cytoplasmic proteins that form a <200-nm plaque linking the ECM to the actin cytoskeleton. The complexity of focal adhesion composition and dynamics implicate an intricate molecular machine. However, focal adhesion molecular architecture remains unknown. Here we used three-dimensional super-resolution fluorescence microscopy (interferometric photoactivated localization microscopy) to map nanoscale protein organization in focal adhesions. Our results reveal that integrins and actin are vertically separated by a ∼40-nm focal adhesion core region consisting of multiple protein-specific strata: a membrane-apposed integrin signalling layer containing integrin cytoplasmic tails, focal adhesion kinase and paxillin; an intermediate force-transduction layer containing talin and vinculin; and an uppermost actin-regulatory layer containing zyxin, vasodilator-stimulated phosphoprotein and α-actinin. By localizing amino- and carboxy-terminally tagged talins, we reveal talin's polarized orientation, indicative of a role in organizing the focal adhesion strata. The composite multilaminar protein architecture provides a molecular blueprint for understanding focal adhesion functions.
- Published
- 2010
- Full Text
- View/download PDF
47. Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure.
- Author
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Shtengel G, Galbraith JA, Galbraith CG, Lippincott-Schwartz J, Gillette JM, Manley S, Sougrat R, Waterman CM, Kanchanawong P, Davidson MW, Fetter RD, and Hess HF
- Subjects
- Animals, Cell Line, Chlorocebus aethiops, Humans, Microtubules, Imaging, Three-Dimensional instrumentation, Imaging, Three-Dimensional methods, Microscopy, Fluorescence instrumentation, Microscopy, Fluorescence methods, Microscopy, Interference instrumentation, Microscopy, Interference methods
- Abstract
Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.
- Published
- 2009
- Full Text
- View/download PDF
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