Challenges Facing an EM Core Laboratory: Mitochondria Structural Preservation and 3DEM Data Presentation

Liang, Feng-Xia; Petzold, Chris; Dancel-Manning, Kristen; Sall, Joseph; Ren, Patrick H.; Zhou, Chuxuan

doi:10.1017/s1551929520001777

Cited by 7 publications

(3 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…[77] This results in a reduction in artifacts compared with traditional EM using chemical fixation. [78] However, high-pressure freezing and freeze substitution remain resource-intensive, [79] making techniques to minimize artifacts in chemical fixation, the most commonly utilized fixation method, still relevant. [80] Along with the development of the optimized fixation protocol, we described multiple methods to assess mitochondrial morphology.…”

Section: Discussionmentioning

confidence: 99%

A Comprehensive Approach to Sample Preparation for Electron Microscopy and the Assessment of Mitochondrial Morphology in Tissue and Cultured Cells

et al. 2023

View full text Add to dashboard Cite

Mitochondria respond to metabolic demands of the cell and to incremental damage, in part, through dynamic structural changes that include fission (fragmentation), fusion (merging of distinct mitochondria), autophagic degradation (mitophagy), and biogenic interactions with the endoplasmic reticulum (ER). High resolution study of mitochondrial structural and functional relationships requires rapid preservation of specimens to reduce technical artifacts coupled with quantitative assessment of mitochondrial architecture. A practical approach for assessing mitochondrial fine structure using two dimensional and three dimensional high‐resolution electron microscopy is presented, and a systematic approach to measure mitochondrial architecture, including volume, length, hyperbranching, cristae morphology, and the number and extent of interaction with the ER is described. These methods are used to assess mitochondrial architecture in cells and tissue with high energy demand, including skeletal muscle cells, mouse brain tissue, and Drosophila muscles. The accuracy of assessment is validated in cells and tissue with deletion of genes involved in mitochondrial dynamics.

show abstract

Section: Discussionmentioning

confidence: 99%

A Comprehensive Approach to Sample Preparation for Electron Microscopy and the Assessment of Mitochondrial Morphology in Tissue and Cultured Cells

et al. 2023

View full text Add to dashboard Cite

show abstract

“…Transmission electron microscopy FACS-sorted naive CD4 + T cells from LNs and spleen were pelleted at 400g for 4 minutes by swinging bucket Eppendorf 5810 into BEEM conical capsule tips (69913-05, Electron Microscopy Sciences), and fixed in 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer (CB; pH 7.2) overnight at 4°C. The sample was processed following the reduced osmium-thiocarbohydrazide-osmium (rOTO) method (69,70). Briefly, after washing with 0.1 M CB three times for 5 minutes each, the cells were postfixed with 2% osmium tetroxide and 1.5% potassium ferrocyanide in 0.1 M CB for 1.5 hours on ice.…”

Section: Treatmentsmentioning

confidence: 99%

S1PR1 inhibition induces proapoptotic signaling in T cells and limits humoral responses within lymph nodes

Dixit,

Hallisey,

Zhu

et al. 2024

Journal of Clinical Investigation

View full text Add to dashboard Cite

Effective immunity requires a large, diverse naive T cell repertoire circulating among lymphoid organs in search of antigen. Sphingosine 1-phosphate (S1P) and its receptor S1PR1 contribute by both directing T cell migration and supporting T cell survival. Here, we addressed how S1P enables T cell survival and the implications for patients treated with S1PR1 antagonists. We found that S1PR1 limited apoptosis by maintaining the appropriate balance of BCL2 family members via restraint of JNK activity. Interestingly, the same residues of S1PR1 that enable receptor internalization were required to prevent this proapoptotic cascade. Findings in mice were recapitulated in ulcerative colitis patients treated with the S1PR1 antagonist ozanimod, and the loss of naive T cells limited B cell responses. Our findings highlighted an effect of S1PR1 antagonists on the ability to mount immune responses within lymph nodes, beyond their effect on lymph node egress, and suggested both limitations and additional uses of this important class of drugs.

show abstract

“…FACS-sorted naïve CD4+ T cells from LNs and spleen were pelleted at 400 g for 4 mins by swinging bucket Eppendorf 5810 into BEEM conical capsule tips (EMS, #69913-05), and fixed in 2% glutaraldehyde and 2% paraformaldehyde in 0.1M sodium cacodylate buffer (CB, pH 7.2) overnight at 4°C. The sample was processed following rOTO method (69,70). In brief, after washing with 0.1M CB three times for 5 min each, the cells were post-fixed with 2% osmium tetroxide and 1.5% potassium ferrocyanide in 0.1M CB for 1.5 hours on ice.…”

Section: Transmission Electron Microscopymentioning

confidence: 99%

Sphingosine 1-phosphate receptor 1 inhibition induces a pro-apoptotic signaling cascade in T cells

Dixit,

Hallisey,

Zhu

et al. 2023

Preprint

View full text Add to dashboard Cite

Effective immunity requires a large, diverse naïve T cell repertoire circulating among lymphoid organs in search of antigen. Sphingosine 1-phosphate (S1P) and its receptor S1PR1 contribute by both directing T cell migration and supporting T cell survival. Here, we address how S1P enables T cell survival, and the implications for patients treated with S1PR1 antagonists. Contrary to expectations, we found that S1PR1 limits apoptosis by maintaining the appropriate balance of BCL2 family members via restraint of JNK activity. Interestingly, the same residues of S1PR1 that enable receptor internalization are required to prevent this pro-apoptotic cascade. Findings in mice were recapitulated in ulcerative colitis patients treated with the S1PR1 antagonist ozanimod, and the loss of naïve T cells limited B cell responses. Our findings highlight an unexpected effect of S1PR1 antagonists on the ability to mount immune responses within lymph nodes, beyond their effect on lymph node egress, and suggest both limitations and novel uses of this important class of drugs.

show abstract

Challenges Facing an EM Core Laboratory: Mitochondria Structural Preservation and 3DEM Data Presentation

Abstract: Abstract

Cited by 7 publications

References 26 publications

A Comprehensive Approach to Sample Preparation for Electron Microscopy and the Assessment of Mitochondrial Morphology in Tissue and Cultured Cells

A Comprehensive Approach to Sample Preparation for Electron Microscopy and the Assessment of Mitochondrial Morphology in Tissue and Cultured Cells

S1PR1 inhibition induces proapoptotic signaling in T cells and limits humoral responses within lymph nodes

Sphingosine 1-phosphate receptor 1 inhibition induces a pro-apoptotic signaling cascade in T cells

Contact Info

Product

Resources

About