Palladin Can Compensate for Arp2/3 Complex Defects and Structurally Organizes Actin‐Rich Structures Generated during <i>Listeria monocytogenes</i> Infections
Bibliographic record
Abstract
The actin cytoskeleton is co‐opted by the invasive and motile bacteria Listeria monocytogenes ( Listeria ) for their entry, intracellular motility and dissemination from one cell to another. Once inside host cells, these bacteria are propelled by bacterially‐generated, Arp2/3‐based, actin‐rich structures called comet tails. The host protein palladin is a crucial actin crosslinker of motile actin‐based structures generated during eukaryotic cell motility. Recently, palladin has been shown to nucleate branched actin filament arrays in vitro . Whether this new actin‐nucleating role of palladin is involved in powering cell motility remains unknown. Here we set out to test the hypothesis that palladin is a crucial component of bacterially‐induced actin‐rich structures generated during Listeria infections and simultaneously used Listeria as a model to uncover new physiological functions of palladin. Through immunofluorescence microscopy we initially identified palladin at sites of bacterial entry and at motile comet tails. Infections of epithelial cells depleted of palladin through RNAi revealed that palladin was dispensable for bacterial invasion. However, when comet tails were examined, we found that they were significantly shorter and severely misshapen in the absence of palladin. Furthermore, when palladin mutants defective for actin or VASP binding were overexpressed, comet tails either disintegrated at their distal ends or became progressively thinner as they moved, suggesting that palladin is a crucial organizer of the actin arrays within comet tails. Using electron microscopy, we further demonstrated that comet tail thinning resulted in switching of the actin network from branched actin arrays to parallel actin bundles. Finally, to determine if palladin's actin‐nucleating function could compensate for the Arp2/3 complex during comet tail motility, we overexpressed palladin in cells treated with the Arp2/3 inhibitor CK‐666. Remarkably, in the presence of CK‐666, Listeria could initiate and maintain comet tail motility in palladin overexpressing cells, whereas Listeria motility immediately ceased in cells with endogenous levels of palladin. Taken together we show that palladin is essential for normal comet tail function as well as morphology, and when overexpressed, can compensate for Arp2/3 complex functional defects during bacterial actin‐based motility. Support or Funding Information Grant Funding Source: NSERC (grant no. 355316 to J.A.G and grant no. 155397‐2013 to A.W.V), NIH and SFU departmental funds
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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 | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.001 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.003 | 0.001 |
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".