A Versatile Micromanipulation Apparatus for Biophysical Assays of the Cell Nucleus

Currey, Marilena L.; Kandula, Viswajit; Biggs, Ronald; Marko, John F.; Stephens, Andrew D.

doi:10.1007/s12195-022-00734-y

Cited by 9 publications

(8 citation statements)

References 42 publications

(50 reference statements)

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“…However, loss of facultative heterochromatin alone does not match the high levels of nuclear blebbing and rupture in LMNB1−/−, suggesting loss of lamin B1 causes an additive effect. To ultimately determine if lamin B1 is a chromatin perturbation, we would need to conduct micromanipulation force measurements to assay the separate short-extension chromatin-based regime vs. long-extension lamin-based regime (Stephens et al ., 2017; Currey et al ., 2022). Lamin B1’s role in nuclear mechanics, morphology, and compartmentalization remains to be settled, but our data clearly provide evidence that changes to histone modification state are a major contributor to its phenotype.…”

Section: Discussionmentioning

confidence: 99%

Actin contraction controls nuclear blebbing and rupture independent of actin confinement

Pho

Berrada

Gunda

et al. 2022

Preprint

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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.

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Section: Discussionmentioning

confidence: 99%

Actin contraction controls nuclear blebbing and rupture independent of actin confinement

Pho

Berrada

Gunda

et al. 2022

Preprint

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“…To directly measure whether nuclear rigidity is altered by RNA pol II inhibition, we used micropipette micromanipulation of individual nuclei to measure the nuclear force-extension relation (Stephens et al ., 2017; Currey et al ., 2022). Micromanipulation force measurements uniquely provide the ability to measure both short-extension chromatin-based nuclear stiffness and lamin-A-based strain stiffening at longer extensions (>3 μm).…”

Section: Resultsmentioning

confidence: 99%

“…As first described (Stephens et al ., 2017) and more recently updated (Currey et al ., 2022), MEF vimentin null (V-/-) cells were grown in a micromanipulation well to provide low angle access via micropipettes. MEF V−/− nuclei were isolated from living cells via spray micropipette of mild detergent Triton X-100 0.05% in PBS.…”

Section: Methodsmentioning

confidence: 99%

“…Chromatin mechanics is essential to nuclear mechanical response, and it is a key determinant of nuclear shape and stability (Stephens et al ., 2019b). Chromatin generates a spring-like elastic response to small, few-microns-sized mechanical strains, complementing the robust elastic response to large deformations provided by lamins (Stephens et al ., 2017; Hobson et al ., 2020; Currey et al ., 2022). The rigidity of chromatin is governed by histone modifications (Chalut et al ., 2012; Krause et al ., 2013; Shimamoto et al ., 2017; Stephens et al ., 2018, 2019a; Hobson et al .,2020; Nava et al ., 2020), H1 dynamics (Furusawa et al ., 2015; Senigagliesi et al ., 2019), chromatin-binding proteins (Schreiner et al ., 2015; Wang et al ., 2018; Williams et al ., 2020; Strom et al ., 2021), and 3D genome organization (Belaghzal et al ., 2021).…”

Section: Introductionmentioning

confidence: 99%

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Transcription regulates bleb formation and stability independent of nuclear rigidity

Berg

Currey

Gupta

et al. 2022

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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.

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“…Beyond this, tetraploid cells result in larger nuclei. It is also possible that tetraploid cells could have an abnormal nuclear mechanics via chromatin’s contribution (Stephens et al 2017; Banigan et al 2017; Hobson et al 2020; Currey et al 2022) that would disrupt many other important mechanisms in nuclear mechanobiology including mechanotransduction (Kalukula et al 2022).…”

Section: Discussionmentioning

confidence: 99%

CTCF is essential for proper mitotic spindle structure and anaphase segregation

Chiu

Berrada

Eskndir

et al. 2023

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

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A Versatile Micromanipulation Apparatus for Biophysical Assays of the Cell Nucleus

Cited by 9 publications

References 42 publications

Actin contraction controls nuclear blebbing and rupture independent of actin confinement

Actin contraction controls nuclear blebbing and rupture independent of actin confinement

Transcription regulates bleb formation and stability independent of nuclear rigidity

CTCF is essential for proper mitotic spindle structure and anaphase segregation

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