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628 results on '"Carter, S. D."'

20. Density distribution of peripheral blood lymphocytes in health and rheumatoid disease

Author

Carter, S. D.

Subjects
616.7
Abstract

The incidence, nature and significance of activated lymphocytes has been investigated in P.A. and healthy subjects. The incidence of these cells using discontinuous Ficoll gradient analysis was studied in R.A. patients with different disease activity and in those undergoing gold therapy. The density gradient also provided a method for separating lymphocyte subpopulations for the further study of surface markers. These included the possession of surface Ig and receptors for sheep erythrocytes and IgG (Fc). R.A. patients, particularly those with clinically active disease, had more circulatory activated lymphocytes (immunoblasts) and low density lymphocytes than healthy subjects. This increase in low density cells was not seen in patients receiving gold therapy. Surface marker analysis showed that the population of low density (activated) lymphocytes in R.A. were enriched in surface Ig bearing cells and depleted of cells with sheep erythrocyte receptors, in comparison with the high density (small) lymphocytes. However, by comparison, high avidity Fc receptors were found predominantly on the dense lymphocytes whereas low avidity Fc receptors were more common on the low density cells. The role of the activated cells ir R.A. is considered with particular reference to the increased B cell activation in peripheral blood. The propagation of R.A. by activated lymphocytes is discussed in relation to their abnormal migratory patterns to inflammatory sites.

Published
1979

43. The activated endoplasmic reticulum stress sensor IRE1 oligomerizes into filaments contained in 30 nm membrane tubes of complex topology

Author

Tran, N. T., Carter, S. D., Belyy, V., Acosta-Alvear, D., Jensen, G. J., and Walter, P.

Abstract

The unfolded protein response (UPR) is an intracellular signaling network that adjusts the abundance and protein folding capacity of the endoplasmic reticulum (ER) according to need. The most conversed branch of the UPR is mediated by the ER‐resident transmembrane kinase/endoribonuclease IRE1. It senses unfolded protein accumulation within the ER and transduces the signal via a non‐conventional mRNA splicing mechanism. In response to direct binding of unfolded proteins in the ER lumen, IRE1 activates by oligomerization and accumulates in dynamic foci. IRE1 foci are not autophagosomes as they did not colocalize with the autophagosomal marker LC3. Fluorescence recovery after photobleaching (FRAP) experiments indicate that IRE1 molecules in the foci remain in equilibrium with IRE1 molecules in the surrounding ER network. We determined the structure of IRE1 foci in cells by whole cell correlative light – electron tomography. Our results show that IRE1 oligomers induce membrane deformations, leading to the protrusion of narrow 30 nm ribosome‐free tubes that remain connected to the ER and are twisted into glomeruli of complex topology. The tubes contain two parallel filaments in their lumen, likely representing oligomerized IRE1 ER‐lumenal domains. Taken together, our findings define a previously unrecognized subdomain of the ER membrane and shed new light on the structure and organization of active mammalian IRE1 inside the cell.

Published
2017

44. Development of cryogenic correlated light electron microscopy methods to study mechanisms of intracellular trafficking and their relationships to the secretory pathway

Author

Carter, S. D., Mageswaran, S. K., Farino, Z. J., Mamede, J. I., Hope, T. J., Frank, J., Freyberg, Z., and Jensen, G. J.

Abstract

The application of cryogenic electron microscopy (cryo‐EM) to the study of cellular ultrastructure provides a resolution several orders of magnitude better than light microscopy. Although this approach is increasingly applied in situ, it suffers from limitations in our ability to target imaging to specific intracellular features including the subcellular localization of specific events of interest. Cryogenic correlated light and electron microscopy (cryo‐CLEM) helps to overcome this problem by spatially locating areas of interest inside cells using fluorescence from genetically tagged or stained cellular molecules and allows for the visualization of localized fluorescently‐tagged proteins down to the level of individual organelles. Here, we attempted to study the secretory pathway in a specialized mammalian cell line, insulin‐secreting INS‐1E cells, expressing genetically‐encoded fluorophores as a model system to develop a cryo‐CLEM methodology. We discovered that there are many bright sources of autofluorescence in frozen cells. Based on our initial observations and the current understanding in the field, we hypothesized that autofluorescence from endogenous cellular substrates exhibits a broader spectrum of fluorescence than the fluorescence range of our expressed fluorescent proteins. To test this, we developed a quantitative approach to discriminate between autofluorescence and the fluorescent signal from genetically‐encoded fluorophores by measuring fluorescent intensities across different bandwidths. To validate this new methodology, we visualized multiple fluorophore‐tagged organelle markers in our experimental cell system. We found that DsRed2‐cytochrome c oxidase and chromogranin A‐GFP proteins were targeted in INS‐1E cells to mitochondria and secretory granules by cryo‐CLEM, consistent with their respective well‐established intracellular localizations. Moreover, these fluorescent signals were clearly distinguishable from autofluorescence emanating from endogenous structures including insulin crystals and multilamellar bodies. Overall, our novel cryo‐CLEM methods open the door to the study of cellular phenomena and structures with a new degree of specificity.

Published
2016

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