A high-content screen reveals new regulators of nuclear membrane stability DOI Creative Commons
Amanda Gunn, Artem I. Yashchenko, Julien Dubrulle

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: May 31, 2023

Nuclear membrane rupture is a physiological response to multiple in vivo processes, such as cell migration, that can cause extensive genome instability and upregulate invasive inflammatory pathways. However, the underlying molecular mechanisms of are unclear few regulators have been identified. In this study, we developed reporter size excluded from re-compartmentalization following nuclear events. This allows for robust detection factors influencing integrity fixed cells. We combined with an automated image analysis pipeline high-content siRNA screen identify new proteins both increase decrease frequency cancer Pathway identified enrichment ER our hits demonstrate one these, protein phosphatase CTDNEP1, required stability. Further known contributors, including newly quantitative lamina gaps, strongly suggests CTDNEP1 acts pathway. Our findings provide insights into mechanism define highly adaptable program removes substantial barrier discoveries field.

Language: Английский

CTCF is essential for proper mitotic spindle structure and anaphase segregation DOI Creative Commons

Katherine Chiu,

Yasmin Berrada,

Nebiyat Eskndir

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Jan. 10, 2023

Mitosis is an essential process in which the duplicated genome segregated equally into two daughter cells. CTCF has been reported to be present mitosis but its importance for mitotic fidelity remains determined. To evaluate of mitosis, we tracked behaviors wild type and different CRISPR-based genetic knockdowns. We find that knockdown results prolonged mitoses failed anaphase segregation via time lapse imaging SiR-DNA. did not alter cell cycling or checkpoint, was activated upon nocodazole treatment. Immunofluorescence spindle knockdowns revealed disorganization tri/tetrapolar spindles chromosomes behind pole. Imaging interphase nuclei showed nuclear size increased drastically, consistent with failure divide anaphase. Population measurements shape do display decreased circularity blebbing relative type. However, abnormal morphologies successful mitoses, suggesting population images capture individual behaviors. Thus, important both proper metaphase organization impacts nucleus.

Language: Английский

Citations

3
Cell Morphology accurately predicts the nuclear shape of adherent cells DOI Creative Commons

Sebastian Lawton,

Rosaline A. Danzman,

Rocco Spagnuolo

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 28, 2024

Summary Cells are internally tensed, or prestressed, largely by actomyosin contractility. We hypothesized that nuclear shape is quantitatively predictable from cell since prestress couples them both. trained machine learning models on a publicly available image database of the WTC-11 line and predicted modes nucleus with high accuracy. develop U-Net architecture-based model, Cell2Nuc, voxels membrane accuracies between 74%-87%. To investigate prestress, we cultured imaged HeLa cells after inhibiting The Cell2Nuc model retrained slightly lower Statistical analysis revealed changes in size chromatin organization upon inhibition. Similar trends were seen images taken NIH3T3 cells. Thus, encodes features shape, their coupling partly due to contractility, whose abrogation leads mechanosensitive origin.

Language: Английский

Citations

0
Differentiation-dependent chromosomal organization changes in normal myogenic cells are absent in rhabdomyosarcoma cells DOI Creative Commons

Joe Ibarra,

Tyler Hershenhouse,

Luay M. Almassalha

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: May 11, 2023

Myogenesis, the progression of proliferating skeletal myoblasts to terminally differentiated myotubes, regulates thousands target genes. Uninterrupted linear arrays such genes are differentially associated with specific chromosomes, suggesting chromosome regulatory roles in myogenesis. Rhabdomyosarcoma (RMS), a tumor muscle, shares common features normal muscle cells. We hypothesized that RMS and myogenic cells possess differences chromosomal organization related gene arrangement. compared organizational characteristics chromosomes 2 18, chosen for their difference arrangement, cultured cell lines myotubes. found chromosome-specific during myogenesis, increased area occupied shift peripheral localization specifically 2. Most strikingly, we differentiation-dependent positioning relative nuclear axis, preferential along major axis present only demonstrated no preference axial positioning, but induced differentiation through transfection pro-myogenic miRNA miR-206 resulted an increase Our findings identify both differentiation-dependent, change highlight role spatial differentiation.

Language: Английский

Citations

1
Differentiation-dependent chromosomal organization changes in normal myogenic cells are absent in rhabdomyosarcoma cells DOI Creative Commons

Joe Ibarra,

Tyler Hershenhouse,

Luay M. Almassalha

et al.

Frontiers in Cell and Developmental Biology, Journal Year: 2023, Volume and Issue: 11

Published: Nov. 7, 2023

Myogenesis, the progression of proliferating skeletal myoblasts to terminally differentiated myotubes, regulates thousands target genes. Uninterrupted linear arrays such genes are differentially associated with specific chromosomes, suggesting chromosome regulatory roles in myogenesis. Rhabdomyosarcoma (RMS), a tumor muscle, shares common features normal muscle cells. We hypothesized that RMS and myogenic cells possess differences chromosomal organization related gene arrangement. compared organizational characteristics chromosomes 2 18, chosen for their difference arrangement, cultured cell lines myotubes. found chromosome-specific during myogenesis, increased area occupied shift peripheral localization specifically 2. Most strikingly, we differentiation-dependent positioning relative nuclear axis, preferential along major axis present only demonstrated no preference axial positioning, but induced differentiation through transfection pro-myogenic miRNA miR-206 resulted an increase Our findings identify both differentiation-dependent, change highlight role spatial differentiation.

Language: Английский

Citations

1
A high-content screen reveals new regulators of nuclear membrane stability DOI Creative Commons
Amanda Gunn, Artem I. Yashchenko, Julien Dubrulle

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: May 31, 2023

Nuclear membrane rupture is a physiological response to multiple in vivo processes, such as cell migration, that can cause extensive genome instability and upregulate invasive inflammatory pathways. However, the underlying molecular mechanisms of are unclear few regulators have been identified. In this study, we developed reporter size excluded from re-compartmentalization following nuclear events. This allows for robust detection factors influencing integrity fixed cells. We combined with an automated image analysis pipeline high-content siRNA screen identify new proteins both increase decrease frequency cancer Pathway identified enrichment ER our hits demonstrate one these, protein phosphatase CTDNEP1, required stability. Further known contributors, including newly quantitative lamina gaps, strongly suggests CTDNEP1 acts pathway. Our findings provide insights into mechanism define highly adaptable program removes substantial barrier discoveries field.

Language: Английский

Citations

0