Biology subjects

Pfeifer, C. R.

Publications and source records attributed to Pfeifer, C. R..

4 recordsLinked to original sources

bioRxiv2021

Gaussian curvature dilutes the nuclear lamina, favoring nuclear rupture, especially at high strain rate

Nuclear rupture has long been associated with deficits or defects in lamins, with recent results also indicating a role for actomyosin stress, but key physical determinants of rupture remain unclear. Here, lamin-B stably interacts with the nuclear membrane at sites of low Gaussian curvature yet dilutes at high-curvature to favor rupture, whereas lamin-A depletes similarly but only at high strain-rates. Live cell imaging of lamin-B1 gene-edited cancer cells is complemented by fixed-cell imaging of ruptured nuclei in: iPS-derived cells from progeria patients, cells within beating chick embryo hearts, and cancer cells that develop multiple ruptures in migrating through small pores. Dilution and curvature-dependent rupture fit a parsimonious model of a stiff filament that detaches from a curved surface, suggesting an elastic-type response of lamin-B, but rupture is also modestly suppressed by inhibiting myosin-II and by hypotonic stress, which slow the strain rates. Lamin-A dilution and nuclear rupture likelihood indeed increase above a threshold rate of pulling into small pipettes, suggesting a viscoplastic coupling to the envelope for protection against nuclear rupture. Summary statementHigh nuclear curvature drives lamina dilution and nuclear envelope rupture even when myosin stress is inhibited. Stiff filaments generally dilute from sites of high Gaussian curvature, providing mathematical fits of experiments.

biophysics↗

bioRxiv2018

Live cell monitoring for factors affecting genome variation

Cancer cells and pluripotent stem cells frequently exhibit gains or losses of entire chromosomes and chromosome segments, and the typical terminal analyses of genomes suggest this aneuploidy is ongoing and particularly variable in solid tumors. Here, we quantify aneuploidy-inducing perturbations by live cell fluorescence monitoring for changes in chromosome-5 in a lung cancer line and in normal diploid iPS cells. Inhibition of the spindle assembly checkpoint (SAC) and knockdown of DNA repair factors cause chromosome mis-segregation to increase several-fold above a low baseline level, and both perturbations also generate several-fold more rare fluorescent-null cells. Loss of chromosome-5 is confirmed by single cell karyotyping, SNP arrays on stable isolated clones, and downregulated expression of genes on chromosome-5 in single cell transcriptomics. The iPS cells also show loss of fluorescence in infrequent cells after SAC inhibition and upon growth as teratomas in mice. Viability, selection, and altered expression can thus be tracked to reveal molecular mechanisms in aneuploidy.

genomics↗

bioRxiv2018

Rescue of DNA damage in cells after constricted migration reveals bimodal mechano-regulation of cell cycle

Migration through constrictions can clearly rupture nuclei and mis-localize nuclear proteins but damage to DNA remains uncertain as does any effect on cell cycle. Here, myosin-II inhibition rescues rupture and partially rescues the DNA damage marker {gamma}H2AX, but an apparent delay in cell cycle is unaffected. Co-overexpression of multiple DNA repair factors and antioxidant inhibition of break formation also have partial effects, independent of rupture. However, there seems to be a bimodal dependence of cell cycle on DNA damage. Migration through custom-etched pores yields the same bimodal, with ~4-m pores causing intermediate levels of damage and cell cycle delay. Micronuclei (generated in faulty division) of the smallest diameter appear similar to ruptured nuclei, with high DNA damage and entry of chromatin-binding cGAS (cyclic-GMP-AMP-synthase) from cytoplasm but low repair factor levels. Increased genomic variation after constricted migration is quantified in expanding clones and is consistent with (mis)repair of excess DNA damage and subsequent proliferation.

cell biology↗

bioRxiv2017

Cell cycle repression and DNA repair defects follow constricted migration

Cancer cell invasion into tissue or narrow capillaries often elongates the nucleus and sometimes damages it, but cell cycle effects are unknown and highly relevant to tumorigenesis. Here, nuclear rupture and DNA breaks caused by constricted migration are quantified in different phases of cell cycle - which is effectively repressed. Cancer lines with varying levels of contact inhibition and lamina proteins exhibit diverse frequencies of nuclear lamina rupture after migration, with prerupture dilation of gene-edited RFP-Lamin-B1 preceding DNA repair factor leakage in pressure-controlled distension. Post-migration rupture indeed associates with mis-localized DNA repair factors and increased DNA breaks as quantified by pan-nucleoplasmic foci of {gamma}H2AX, with foci counts always suppressed in late cell cycle. When contact-inhibited cells migrate through large pores into sparse microenvironments, cells re-enter cell cycle consistent with release from contact inhibition. In contrast, constricting pores effectively delay re-entry, but the excess DNA damage nonetheless exceeds any cell cycle dependence. Partial depletion of topoisomerase does not strongly affect cell cycle or the excess DNA damage, consistent with weak dependencies on replication stress. Constricted migration thus impacts cell cycle as well as DNA damage.

biophysics↗