Biology subjects

Pham, N. P.

Publications and source records attributed to Pham, N. P..

3 recordsLinked to original sources

bioRxiv2025

The nuclear localization of Ect2 is required for cytokinesis

In cytokinesis, a contractile ring constricts the cell to form two daughters. After ingression, the ring matures into the midbody, and an intercellular bridge connects the daughter cells until it is cut during abscission. Ring assembly requires the activation of RhoA GTPase at the equatorial cortex by Ect2, a guanine nucleotide exchange factor (GEF). However, it is unclear if RhoA must be inactivated after ring closure to complete cytokinesis. Here, we show that the nuclear sequestration of Ect2 after ingression is required for cytokinesis. Mutating the nuclear localization signal (NLS) in Ect2 causes it to remain at the midbody where it generates persistent active RhoA, leading to cytokinesis failure. Re-localizing the mutant to the nucleus using SV40NLS restores cytokinesis. Further, over-expression of mutant Ect2 causes cytokinesis failure, which is rescued by reducing GEF activity. We propose that persistent RhoA causes instability in the intercellular bridge, preventing abscission. Nuclear sequestration may regulate the function of other contractile proteins with NLS sequences.

cell biology↗

bioRxiv2023

Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies

Endogenous tags have become invaluable tools to visualize and study native proteins in live cells. However, generating human cell lines carrying endogenous tags is difficult due to the low efficiency of homology-directed repair. Recently, an engineered split mNeonGreen protein was used to generate a large-scale endogenous tag library in HEK293 cells. Using split mNeonGreen for large-scale endogenous tagging in human iPSCs would open the door to studying protein function in healthy cells and across differentiated cell types. We engineered an iPS cell line to express the large fragment of the split mNeonGreen protein (mNG21-10) and showed that it enables fast and efficient endogenous tagging of proteins with the short fragment (mNG211). We also demonstrate that neural network-based image restoration enables live imaging studies of highly dynamic cellular processes such as cytokinesis in iPSCs. This work represents the first step towards a genome-wide endogenous tag library in human stem cells.

cell biology↗

bioRxiv2019

Intramolecular regulation of anillin during cytokinesis

Cytokinesis occurs by the ingression of an actomyosin ring that cleaves a cell into two daughters. This process is tightly controlled to avoid aneuploidy, and we previously showed that active Ran coordinates ring positioning with chromatin. Active Ran is high around chromatin, and forms an inverse gradient to cargo-bound importins. We found that the ring component anillin contains an NLS that binds to importin and is required for its function. Anillin contains a RhoA-binding domain (RBD), which we revealed autoinhibits the adjacent NLS-containing C2 domain. Here, we show that active RhoA relieves inhibition of the C2 domain. Furthermore, FRAP experiments show that the NLS regulates anillins cortical properties, supporting feedback to the RBD. Indeed, mutations that disrupt the interface between the RBD and C2 domain disrupt anillins localization and function. Thus, active RhoA induces a conformational change that increases accessibility to the C2 domain, which is maintained by importin-binding for recruitment to the equatorial cortex.

cell biology↗