Whole cell and bacterial movement: The identification of the ubiquitin E2 enzyme (Ube2N) as a novel actin‐associated protein
Bibliographic record
Abstract
Actin dynamics are tightly regulated at the leading edge of motile cells. At that region of the cell, the plasma membrane flattens into a structure called the lamellipodium where an assortment of proteins is recruited to polymerize actin filaments. Extension of the lamellipodium relies on the power generated by the actin filaments to move the plasma membrane forward. To characterize the proteins involved in whole cell motility, researchers often use bacterial pathogens that hijack the eukaryotic cell actin cytoskeleton. One model system utilizes Listeria monocytogenes . During these infections, the bacteria control actin polymerizing proteins at various stages as the infections progress, 1) during their invasion, 2) as they form actin clouds around the microbes, 3) when comet tails are generated to enable the bacteria to move within the host cells and 4) at modified comet tails (called listeriopods) used to transfer the bacteria from one cell to another. Using these infections, we found that the ubiquitin conjugating enzyme Ube2N was recruited to all sites of actin concentration generated by the bacteria, but was very heavily concentrated at listeriopods. Since Ube2N was recruited to actin structures especially at the plasma membrane, we hypothesized that Ube2N may play a role in regulating actin dynamics involved in in cell motility. To test this hypothesis, we determined if Ube2N was required for actin‐based internalization of L. monocytogenes and listeriopod generation during bacterial cell‐to‐cell spread. Using a Ube2N‐specific inhibitor (NSC697923) to block Ube2N function in host cells, we showed that bacterial internalization was reduced by 90%. When Ube2N was inhibited after invasion had already occurred, we observed a striking decrease in the proportion of listeriopods resulting in a 40% decrease in the area of bacterial spreading within the monolayer of Ube2N‐inhibited cells. These results indicated that Ube2N function was required for actin dynamics at the plasma membrane. We then immunoprecipitated Ube2N from L. monocytogenes ‐infected cells to identify binding partners of Ube2N and we found that actin was co‐immunoprecipitated with Ube2N. Through far western blotting, we then determined that there was a direct interaction of Ube2N with actin itself and when coupled with subcellular fractionation and immunolocalization we demonstrated that the Ube2N‐actin interaction occurs at the plasma membrane. Finally, to determine if Ube2N was important for whole cell motility, we performed wound healing assays using NSC697923‐treated cells and compared them to control (DMSO)‐treated cells. Wound closure did not occur in NSC697923‐treated cells and there was no significant displacement of individual cells. Moreover, the immunolocalization of Ube2N in these inhibited cells showed that the protein concentrated to the edge of the lamellipodia instead of the diffuse localization pattern in control cells and this suggested that Ube2N regulates actin during cell motility. These results demonstrated that Ube2N is crucial for actin dynamics at the plasma membrane, and is necessary for actin‐based cell motility. Support or Funding Information This study was funded through NSERC and CIHR. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
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 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 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 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".