Biology subjects

Pho, M.

Publications and source records attributed to Pho, M..

5 recordsLinked to original sources

bioRxiv2025

Changes in nuclear and actin mechanics from G1 to G2 affect nuclear integrity

The structural integrity of the nucleus is dependent on nuclear mechanical elements of chromatin and lamins to resist antagonistic actin cytoskeleton forces. Imbalance results in nuclear blebbing, rupture, and cellular dysfunction found in many human diseases. We used Fluorescent Ubiquitin Cell Cycle Indicator (FUCCI) cells to determine how cell cycle changes affect the nucleus and actin force balance. While nuclear blebs are present equally throughout interphase, nuclear blebs form predominantly in G1 and then persist into G2 due to increased actin-based nuclear confinement and focal adhesion density in G1 vs. G2 cells. Upon artificial confinement, G2 nuclei ruptured more than G1 nuclei. Single nucleus micromanipulation force measurements confirmed that G1 nuclei are stronger than G2 nuclei in both the chromatin-based and lamin-based nuclear stiffness regimes. Decreased nuclear stiffness can be explained by loss of peripheral H3K9me3 from G1 to G2, recapitulated by H3K9me3 inhibition via Chaetocin. Cell cycle-based changes in nuclear and actin mechanics impact nuclear integrity and shape.

cell biology↗

bioRxiv2025

Lamin B loss in nuclear blebs is rupture dependent while increased DNA damage is rupture independent

The nucleus houses genetic information and functions separate from the rest of the cell. Loss of nuclear shape results in nuclear ruptures. Nuclear blebs are deformations identified by decreased DNA density, while lamin B levels vary drastically. To determine if decreased lamin B levels are due to nuclear rupture, we used immunofluorescence to measure levels of lamin B and emerin, a nuclear envelope protein that enriches to sites of nuclear rupture. We observed that cell types that exhibit decreased levels of lamin B also show an enrichment of emerin in nuclear blebs. Oppositely, in other cell types, nuclear blebs display maintained levels of lamin B1 and showed no emerin enrichment. To determine how nuclear rupture affects DNA damage, we time lapse imaged nuclear rupture dynamics then fixed the same cells to conduct immunofluorescence of {gamma}H2AX and emerin. We find that DNA damage levels are higher in blebbed nuclei independent of nuclear rupture. Thus, we confirm that lamin B1 loss in nuclear blebs is due to nuclear rupture and blebbed nuclei have increased DNA damage that is independent of rupture. Summary statement (180-200 characters): We measured lamin B and DNA damage in blebbed nuclei to determine the effect of nuclear rupture. We find that nuclear rupture causes loss of lamin B in nuclear blebs but that increased DNA damage in blebbed nuclei is independent of rupture.

cell biology↗

bioRxiv2024

DNA damage causes ATM-dependent heterochromatin loss leading to nuclear softening, blebbing, and rupture

The nucleus must maintain stiffness to protect the shape and integrity of the nucleus to ensure proper function. Defects in nuclear stiffness caused from chromatin and lamin perturbations produce abnormal nuclear shapes common in aging, heart disease, and cancer. Loss of nuclear shape via protrusions called blebs leads to nuclear rupture that is well-established to cause nuclear dysfunction, including DNA damage. However, it remains unknown how increased DNA damage affects nuclear stiffness, shape, and ruptures, which could create a negative feedback loop. To determine if increased DNA damage alters nuclear physical properties, we treated MEF cells with DNA damage drugs cisplatin and bleomycin. DNA damage drugs caused increased nuclear blebbing and rupture in interphase nuclei within a few hours and independent of mitosis. Micromanipulation force measurements reveal that DNA damage decreased chromatin-based nuclear mechanics but did not change lamin-based strain stiffening at long extensions relative to wild type. Immunofluorescence measurements of DNA damage treatments reveal the mechanism is an ATM-dependent decrease in heterochromatin leading to nuclear weaken, blebbing, and rupture which can be rescued upon ATM inhibition treatment. Thus, DNA damage drugs cause ATM-dependent heterochromatin loss resulting in nuclear softening, blebbing, and rupture.

cell biology↗

bioRxiv2022

Actin contraction controls nuclear blebbing and rupture independent of actin confinement

The nucleus is a mechanically stable compartment of the cell that contains the genome and performs many essential functions. Nuclear mechanical components chromatin and lamins maintain nuclear shape, compartmentalization, and function by resisting antagonistic actin contraction and confinement. However, studies have yet to compare chromatin and lamins perturbations side-by-side as well as modulated actin contraction while holding confinement constant. To accomplish this, we used NLS-GFP to measure nuclear shape and rupture in live cells with chromatin decompaction (VPA), loss of lamin B1 (LMNB1-/-), and loss of lamin A/C (LMNA-/-). We then modulated actin contraction while maintaining actin confinement measured by nuclear height. Wild type, chromatin decompaction, and lamin B1 null present bleb-based nuclear deformations and ruptures dependent on actin contraction and independent of actin confinement. Inhibition of actin contraction by Y27632 decreased nuclear blebbing and ruptures to near 0% of cells while activation of actin contraction by CN03 increased the frequency of ruptures by nearly two-fold. However, lamin A/C null results in overall abnormal shape, but similar blebs and ruptures as wild type which were unaffected by actin contraction modulation. Actin contraction control of nuclear shape and ruptures showed that DNA damage levels were more correlated with perturbed nuclear shape than they were with changes in nuclear ruptures. We reveal that lamin B1 is a chromatin decompaction phenotype because using GSK126, which mimics the loss of facultative heterochromatin in lamin B1 null, is sufficient to phenocopy increased nuclear blebbing and ruptures. Furthermore, even though blebs and ruptures in lamin A/C null cells are insensitive to actin contraction, they do have the capacity to form increased levels of nuclear blebs and bleb-based ruptures, shown by treating with VPA. Thus, nuclear bleb formation and bleb-based nuclear ruptures are driven by actin contraction and independent of changes in actin confinement.

cell biology↗

bioRxiv2022

Transcription regulates bleb formation and stability independent of nuclear rigidity

Chromatin is an essential component of nuclear mechanical response and shape that maintains nuclear compartmentalization and function. The biophysical properties of chromatin alter nuclear shape and stability, but little is known about whether or how major genomic functions can impact the integrity of the nucleus. We hypothesized that transcription might affect cell nuclear shape and rupture through its effects on chromatin structure and dynamics. To test this idea, we inhibited transcription with the RNA polymerase II inhibitor alpha-amanitin in wild type cells and perturbed cells that present increased nuclear blebbing. Transcription inhibition suppresses nuclear blebbing for several cell types, nuclear perturbations, and transcription inhibitors. Furthermore, transcription is necessary for robust nuclear bleb formation, bleb stabilization, and bleb-based nuclear ruptures. These morphological effects appear to occur through a novel biophysical pathway, since transcription does not alter either chromatin histone modification state or nuclear rigidity, which typically control nuclear blebbing. We find that active/phosphorylated RNA pol II Ser5, marking transcription initiation, is enriched in nuclear blebs relative to DNA. Thus, transcription initiation is a hallmark of nuclear blebs. Polymer simulations suggest that motor activity within chromatin, such as that of RNA pol II, can generate active forces that deform the nuclear periphery, and that nuclear deformations depend on motor dynamics. Our data provide evidence that the genomic function of transcription impacts nuclear shape stability, and suggests a novel mechanism, separate and distinct from chromatin rigidity, for regulating large-scale nuclear shape and function.

cell biology↗