Measurement of oligomerization states of membrane proteins via spatial fluorescence intensity fluctuation analysis
Bibliographic record
Abstract
The objective of this thesis is the development and use of a novel fluorescence fluctuation technique for determining the oligomeric state of fluorescently labeled proteins in situ via microscopy imaging. High order moment analysis of fluorescence intensity fluctuations from individual confocal laser scanning microscopy (CLSM) images applied to study monomer-oligomer distributions of fluorescently labeled proteins was developed. Using computer simulations and experiments with fluorescent microspheres and organic fluorescent dyes, the detection limits and accuracy of this statistical approach were determined. A series of control experiments were carried out to support the membrane receptor oligomerization studies in this thesis. The methods were then applied to study oligomerization states in various biological systems. Epidermal growth factor receptors (EGFR) play a critical role in cell growth, proliferation and survival. The activation steps of signal transduction pathways are known to involve EGFR oligomerization. Pharmacodynamic studies of ligand-induced clustering of EGF receptors were carried out. Spatial intensity distribution analysis (SpIDA), which accurately measures monomer-dimer distributions, was used to measure the increase in EGFR dimeric population upon addition of EGF ligand. The distribution of aggregates forming in the course of EGFR internalization was evaluated using two-population moment analysis. The findings supported an existing model proposing two distinct receptor internalization pathways. The electrogenic sodium bicarbonate cotransporter NBCe1-A plays an important role in absorbing sodium bicarbonate across the basolateral membrane of the proximal tubule. The fluorescence moment image analysis and SpIDA were applied to study the oligomeric state of NBCe1-A in cultured mammalian cells expressing various mutants of cotransporter. Spatial fluctuation analysis revealed that NBCe1-A existed on the cell membrane predominantly as a monomer and negligibly as higher order oligomers. To measure the oligomerization state of the native cotransporter, samples of rat kidney tissues were prepared and the native NBCe1-A was immunostained using fluorescently labeled primary antibody against the wild type cotransporter. The image analysis showed that NBCe1-A was present on the proximal tubule basolateral membrane in predominantly dimeric and rarely monomeric or higher order oligomeric states. Human immunodeficiency virus (HIV) diverts the cellular ESCRT machinery to promote the release of newly formed virions from host cells. The ATPase, VPS4A, acts at a late stage of ESCRT function. It provides energy for dissociation of ESCRT complexes and membrane abscision. The fluorescence moment analysis of VPS4A-eGFP images revealed the monomeric distribution of the protein in the plasma membrane outside budding sites and formation of two to five VPS4A dodecamers at the budding sites. The combination of the results of VPS4A dynamics studies together with the VPS4A burst size analysis shed light on steps in the HIV lifecycle and release.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.011 | 0.003 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".