Biology subjects

Piekny, A.

Publications and source records attributed to Piekny, A..

8 recordsLinked to original sources

bioRxiv2025

Importin-β1 functions as a chromatin sensor to position the contractile ring for cytokinesis

Cytokinesis, thefinal step of cell division, relies on the ingression of a carefully positioned actomyosin ring. Chromatin-associated Ran-GTP helps position this ring although the mechanism is not clear. We hypothesize that a depleted zone of Ran-GTP between the segregating chromosomes causes importins to become equatorially enriched and promote the recruitment of the scaffold protein anillin. However, the role of importins during anaphase remains unclear. Here, we tested if importins respond to the chromatin-associated Ran-GTP gradient to regulate ring assembly. Using endogenous tagging, live-cell imaging, and optogenetic perturbations, we found that importin-{beta}1 becomes equatorially enriched and is required for cytokinesis in hypotriploid HeLa cells, but not in euploid HCT 116 cells. A predictive model of the Ran-GTP gradient and Ran-free importin-{beta}1, validated experimentally by FLIM-FRET, identified factors that modulate this chromatin sensing pathway including chromatin-to-cell size ratio. Ourfindings suggest that highly aneuploid cancer cells may depend on importin-mediated anillin recruitment, representing a targetable weakness that distinguishes them from diploid cells. Summary blurbImportin enrichment at the equator promotes anillin recruitment during cytokinesis in a ploidy-dependent manner, linking Ran-GTP gradients to cell division. HighlightsO_LIRan-free Importin-{beta}1 becomes enriched between segregating chromosomes in anaphase HeLa cells but not in HCT116 cells. C_LIO_LIImportin-binding is required for anillin localization and function in cytokinesis in HeLa cells but not in HCT 116 cells. C_LIO_LIImportin-{beta}1 disruption at anaphase causes ring oscillation and cytokinesis failure in HeLa cells. C_LIO_LIImportin-{beta}1 is not equatorially enriched and anillin is broader in HeLa cells with a lower chromatin-to-cell-size ratio. C_LIO_LIImportin-{beta}1 is equatorially enriched and anillin is narrower in HCT 116 cells with a higher chromatin-to-cell-size ratio. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=188 HEIGHT=200 SRC="FIGDIR/small/688041v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1545a59org.highwire.dtl.DTLVardef@f7b8edorg.highwire.dtl.DTLVardef@18df715org.highwire.dtl.DTLVardef@15891d5_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

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↗

bioRxiv2022

Imaging tools generated by CRISPR/Cas9 tagging reveal cytokinetic diversity in mammalian cells

Cytokinesis is required to physically separate the daughter cells at the end of mitosis. This process occurs via the ingression of an actomyosin ring that assembles in anaphase and pulls in the overlying plasma membrane as it constricts. Mechanistic studies have uncovered different pathways that regulate the assembly and position of the ring in mammalian cells, but the majority of these studies were done using HeLa cells with overexpressed transgenes, and the relative requirement for these mechanisms among the majority of cell types is not known. Here, we used CRISPR/Cas9 gene editing to endogenously tag cytokinesis proteins, anillin, Ect2 and RhoA, as well as other cellular components, with fluorescent proteins. These tools enabled the visualization of cytokinesis by live imaging to quantitatively study these proteins at endogenous levels. As a proof-of-concept, tagging anillin in multiple mammalian cell lines revealed cytokinetic diversity, which will be useful for studies of how mechanisms controlling cytokinesis vary among cell types. We also successfully tagged multiple cellular components in the same cell line, demonstrating the versatility of these tagging tools.

cell biology↗

bioRxiv2021

The ubiquitous terpene geosmin is a warning chemical

Known as the smell of earth after rain, geosmin is an odorous terpene detectable by humans at picomolar concentrations. Geosmin production is heavily conserved in actinobacteria, myxobacteria, cyanobacteria, and some fungi, but its biological activity is poorly understood. We theorized that geosmin was an aposematic signal used to indicate the unpalatability of toxin-producing microbes, discouraging predation by eukaryotes. Consistent with this hypothesis we found that geosmin and the related terpene 2-methylisoborneol reduced predation of Streptomyces coelicolor and Myxococcus xanthus by the bacteriophagous Caenorhabditis elegans. Predation was restored by the removal of both terpene biosynthetic pathways or deletion of the C. elegans ASE sensory neuron, and resulted in the death of the nematodes. Geosmin itself was non-toxic. This is the first warning chemical to be identified in bacteria or fungi, and suggests molecular signalling affects microbial predator-prey interactions in a manner similar to the well-studied visual markers of poisonous animal prey.

microbiology↗

bioRxiv2021

The Ran pathway uniquely regulates cytokinesis in cells with different fates in the early C. elegans embryo

Cytokinesis occurs at the end of mitosis and occurs due to the ingression of a contractile ring that cleaves the daughter cells. This process is tightly controlled to prevent cell fate changes or aneuploidy, and the core machinery is highly conserved among metazoans. Multiple mechanisms regulate cytokinesis, but their requirement in different cell types is not known. Here, we show that differently fated AB and P1 cells in the early C. elegans embryo have unique cytokinesis kinetics supported by distinct levels and cortical patterning of myosin. Through perturbation of polarity regulators and the generation of stable tetraploid strains, we demonstrate that these differences depend on both cell fate and size. Additionally, these parameters could influence the Ran pathway, which coordinates the contractile ring with chromatin position, and controls cytokinesis differently in AB and P1 cells. Our findings demonstrate the need to consider multiple parameters when modeling ring kinetics.

cell biology↗

bioRxiv2020

Multi-tissue patterning drives anterior morphogenesis of the C. elegans embryo

Complex structures derived from multiple tissue types are challenging to study in vivo, and our knowledge of how cells from different tissues are coordinated is limited. Model organisms have proven invaluable for improving our understanding of how chemical and mechanical cues between cells from two different tissues can govern specific morphogenetic events. Here we used Caenorhabditis elegans as a model system to show how cells from three different tissues are coordinated to give rise to the anterior lumen. This poorly understood process has remained a black box for embryonic morphogenesis. Using various microscopy and software approaches, we describe the movements and patterns of epidermal cells, neuroblasts and pharyngeal cells that contribute to lumen formation. The anterior-most pharyngeal cells (arcade cells) may provide the first marker for the location of the future lumen and facilitate the patterning of the surrounding neuroblasts. These neuroblast patterns control the rate of migration of the anterior epidermal cells, whereas the epidermal cells ultimately reinforce and control the position of the future lumen, as they must join with the pharyngeal cells for their epithelialization. Our studies are the first to characterize anterior morphogenesis in C. elegans in detail and should lay the framework for identifying how these different patterns are controlled at the molecular level.

developmental 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↗