Multiscale imaging and quantitative analysis of plasma membrane protein-cortical actin interplay
Bibliographic record
Abstract
The spatiotemporal organization of cell surface receptors is important for cell signaling. Cortical actin (CA), the subset of the actin cytoskeleton subjacent to the plasma membrane (PM), plays a large role in cell surface receptor organization. This was however shown largely through actin perturbation experiments, which raise concerns of nonspecific effects and preclude quantification of actin architecture and dynamics under unperturbed conditions. These limitations make it challenging to predict how changes in CA properties can affect receptor organization. To derive direct relationships between the architecture and dynamics of CA and the spatiotemporal organization of PM proteins, including cell surface receptors, we developed a multiscale imaging and computational analysis framework based on the integration of single-molecule imaging (SMI) of PM proteins and fluorescent speckle microscopy (FSM) of CA (combined: SMI-FSM) in the same live cell. SMI-FSM revealed differential relationships between PM proteins and CA based on the PM proteins’ actin binding ability, diffusion type and local CA density. It also highlighted the complexity of cell wide actin perturbation, where we found that global changes in actin properties caused by perturbation were not necessarily reflected in the CA properties near PM proteins, and the changes in PM protein properties upon perturbation varied based on the local CA environment. Given the widespread use of SMI as a method to study the spatiotemporal organization of PM proteins and the versatility of SMI-FSM, we expect it to be widely applicable to enable future investigation of the influence of CA architecture and dynamics on different PM proteins, especially in the context of actin-dependent cellular processes, such as cell migration. Significance: Plasma membrane protein organization, an important factor for shaping cellular behaviors, is influenced by cortical actin, the subset of the actin cytoskeleton near the plasma membrane. Yet it is challenging to directly and quantitatively probe this influence. Here, we developed an imaging and analysis approach that combines single-molecule imaging, fluorescent speckle microscopy and computational statistical analysis to characterize and correlate the spatiotemporal organization of plasma membrane proteins and cortical actin. Our approach revealed different relationships between different proteins and cortical actin, and highlighted the complexity of interpreting cell wide actin perturbation experiments. We expect this approach to be widely used to study the influence of cortical actin on different plasma membrane components, especially in actin-dependent processes.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.001 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.001 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
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 teacher head, 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".