Rescue of DNA damage after constricted migration reveals a mechano-regulated threshold for cell cycle.

Migration through 3D constrictions can cause nuclear rupture and mislocalization of 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 γH2AX, but an apparent block in cell cycle appears unaffected. Co-overexpression of multiple DNA repair factors or antioxidant inhibition of break formation also exert partial effects, independently of rupture. Combined treatments completely rescue cell cycle suppression by DNA damage, revealing a sigmoidal dependence of cell cycle on excess DNA damage. Migration through custom-etched pores yields the same damage threshold, with ∼4-µm pores causing intermediate levels of both damage and cell cycle suppression. High curvature imposed rapidly by pores or probes or else by small micronuclei consistently associates nuclear rupture with dilution of stiff lamin-B filaments, loss of repair factors, and entry from cytoplasm of chromatin-binding cGAS (cyclic GMP-AMP synthase). The cell cycle block caused by constricted migration is nonetheless reversible, with a potential for DNA misrepair and genome variation.

Poros Nucleares y Función Celular en Epitelio Mamario / Nuclear Pores and Cellular Function

El presente artículo tiene como objetivo central evidenciar la interesante relación que se establece entre la función celular y el número de poros nucleares, relación que modula el activo intercambio nucleo-citoplasmatico en distintas etapas del ciclo celular de la estirpe HC11.

The main objective of this article is related to the study of different existing relationships between cellular function and the number of nuclear pores in order to explain the amount of nuclear-cytoplasmatic exchange through HC11 cell cycle stages.

Effects of ovariectomy on the secretory apparatus in the right atrial cardiomyocytes of middle-aged mice.

OBJECTIVE: The aim of the present study was to evaluate the effects of ovariectomy on the secretory apparatus of natriuretic peptides in right atrial cardiomyocytes. METHODS: Nine-month-old mice underwent bilateral ovariectomy or sham surgery. The blood exam of the ovariectomized mice showed results consistent with castrated females. Systolic blood pressure was measured after ovariectomy (9 mo of age) and at the moment of sacrifice (12 mo of age). Fragments of the right atrium were collected and prepared for electron microscopy examination. The following variables were quantified: the quantitative density and area of the natriuretic peptide granules, the relative volume of euchromatin in the nucleus, the number of pores per 10 µm of the nuclear membrane and the relative volumes of the mitochondria and Golgi complex. RESULTS: The cardiomyocytes obtained from ovariectomized mice indicated that the quantitative density and the area of secretory granules of natriuretic peptides were significantly lower compared with the sham-operated mice. Furthermore, there was a decrease in the relative volume of euchromatin, a lower density of nuclear pores, and lower relative volumes of the mitochondria and Golgi complex in the ovariectomized mice compared with the sham-operated mice. These findings suggest a pool with a low turnover rate, i.e., low synthesis and elimination of natriuretic peptides. CONCLUSION: A lack of estrogen caused hypotrophy of the secretory apparatus in right atrial cardiomyocytes that could explain the weak synthesis of natriuretic peptides in mice. Furthermore, one of the mechanisms of blood pressure control was lost, which may explain, in part, the elevated blood pressure in ovariectomized mice.

Effects of ovariectomy on the secretory apparatus in the right atrial cardiomyocytes of middle-aged mice

OBJECTIVE : The aim of the present study was to evaluate the effects of ovariectomy on the secretory apparatus of natriuretic peptides in right atrial cardiomyocytes. METHODS: Nine-month-old mice underwent bilateral ovariectomy or sham surgery. The blood exam of the ovariectomized mice showed results consistent with castrated females. Systolic blood pressure was measured after ovariectomy (9 mo of age) and at the moment of sacrifice (12 mo of age). Fragments of the right atrium were collected and prepared for electron microscopy examination. The following variables were quantified: the quantitative density and area of the natriuretic peptide granules, the relative volume of euchromatin in the nucleus, the number of pores per 10 μm of the nuclear membrane and the relative volumes of the mitochondria and Golgi complex. RESULTS: The cardiomyocytes obtained from ovariectomized mice indicated that the quantitative density and the area of secretory granules of natriuretic peptides were significantly lower compared with the sham-operated mice. Furthermore, there was a decrease in the relative volume of euchromatin, a lower density of nuclear pores, and lower relative volumes of the mitochondria and Golgi complex in the ovariectomized mice compared with the sham-operated mice. These findings suggest a pool with a low turnover rate, i.e., low synthesis and elimination of natriuretic peptides. CONCLUSION: A lack of estrogen caused hypotrophy of the secretory apparatus in right atrial cardiomyocytes that could explain the weak synthesis of natriuretic peptides in mice. Furthermore, one of the mechanisms of blood pressure control was lost, which may explain, in part, the elevated blood pressure in ovariectomized mice. .

Nuclear pores in luteal cells during pregnancy and after parturition and pup removal in the rat. A freeze-fracture study.

In a previous paper we described a pronounced increase of apoptotic nuclei in rat corpus luteum of pregnancy whose programmed chromatin degeneration was induced by the progesterone antagonist mifepristone. Those observations encouraged us to study the apoptotic nuclear membrane during pregnancy and after parturition and pup removal, by using a freeze-fracture technique which allows us to observe 'en face' the nuclear envelop and also permits nuclear pore counting. This study was complemented with the TUNEL assay (TdT-mediated dUTP nick-end labelling). Changes in nuclear pores during pregnancy begin with an intense reduction in number but still showing an even distribution on the nuclear membrane, never forming aggregations sharply separated from pore-free areas, which are characteristic of other apoptotic models. Electron microscopy of thin-sections shows, coincidently with findings in the freeze-fracture replicas, a moderately irregular aggregation of marginal heterochromatin condensations. After nuclear fragmentation and micronuclear formation, pores behave in the usual manner in other apoptotic models, i.e., mainly showing migrations of nuclear pores toward the chromatin-free areas. The present results support the hypothesis that nuclear pore complexes are dynamic structures, which permit their migration toward nuclear membrane areas devoid of chromatin aggregations that might block the nucleocytoplasmic transport in such areas.

Nuclear pores in luteal cells during pregnancy and after parturition and pup removal in the rat: a freeze-fracture study

In a previous paper we described a pronounced increase of apoptotic nuclei in rat corpus luteum of pregnancy whose programmed chromatin degeneration was induced by the progesterone antagonist mifepristone. Those observations encouraged us to study the apoptotic nuclear membrane during pregnancy and after parturition and pup removal, by using a freeze-fracture technique which allows us to observe en face the nuclear envelop and also permits nuclear pore counting. This study was complemented with the TUNEL assay (TdT-mediated dUTP nick-end labelling). Changes in nuclear pores during pregnancy begin with an intense reduction in number but still showing an even distribution on the nuclear membrane, never forming aggregations sharply separated from pore-free areas, which are characteristic of other apoptotic models. Electron microscopy of thin-sections shows, coincidently with findings in the freeze-fracture replicas, a moderately irregular aggregation of marginal heterochromatin condensations. After nuclear fragmentation and micronuclear formation, pores behave in the usual manner in other apoptotic models, i.e., mainly showing migrations of nuclear pores toward the chromatin-free areas. The present results support the hypothesis that nuclear pore complexes are dynamic structures, which permit their migration toward nuclear membrane areas devoid of chromatin aggregations that might block the nucleocytoplasmic transport in such areas.(AU)

Nuclear pores in luteal cells during pregnancy and after parturition and pup removal in the rat: a freeze-fracture study

In a previous paper we described a pronounced increase of apoptotic nuclei in rat corpus luteum of pregnancy whose programmed chromatin degeneration was induced by the progesterone antagonist mifepristone. Those observations encouraged us to study the apoptotic nuclear membrane during pregnancy and after parturition and pup removal, by using a freeze-fracture technique which allows us to observe 'en face' the nuclear envelop and also permits nuclear pore counting. This study was complemented with the TUNEL assay (TdT-mediated dUTP nick-end labelling). Changes in nuclear pores during pregnancy begin with an intense reduction in number but still showing an even distribution on the nuclear membrane, never forming aggregations sharply separated from pore-free areas, which are characteristic of other apoptotic models. Electron microscopy of thin-sections shows, coincidently with findings in the freeze-fracture replicas, a moderately irregular aggregation of marginal heterochromatin condensations. After nuclear fragmentation and micronuclear formation, pores behave in the usual manner in other apoptotic models, i.e., mainly showing migrations of nuclear pores toward the chromatin-free areas. The present results support the hypothesis that nuclear pore complexes are dynamic structures, which permit their migration toward nuclear membrane areas devoid of chromatin aggregations that might block the nucleocytoplasmic transport in such areas.

Pore-linked filaments in anura spermatocyte nuclei.

Pore-linked filaments were visualized in spreads of anuran spermatocyte nuclei using transmission electron microscope. We used Odontophrynus diplo and tetraploid species having the tetraploid frogs reduced metabolic activities. The filaments with 20-40 nm width are connected to a ring component of the nuclear pore complex with 90-120 nm and extend up to 1 microm (or more) into the nucleus. The filaments are curved and connect single or neighboring pores. The intranuclear filaments are associated with chromatin fibers and related to RNP particles of 20-25 nm and spheroidal structures of 0.5 microm, with variations. The aggregates of several neighboring pores with the filaments are more commonly observed in 4n nuclei. We concluded that the intranuclear filaments may correspond to the fibrillar network described in Xenopus oocyte nucleus being probably related to RNA transport. The molecular basis of this RNA remains elusive. Nevertheless, the morphological aspects of the spheroidal structures indicate they could correspond to nucleolar chromatin or to nucleolus-derived structures. We also speculate whether the complex aggregates of neighboring pores with intranuclear filaments may correspond to pore clustering previously described in these tetraploid animals using freeze-etching experiments.

Pore-linked filaments in anura spermatocyte nuclei

Pore-linked filaments were visualized in spreads of anuran spermatocyte nuclei using transmission electron microscope. We used Odontophrynus diplo and tetraploid species having the tetraploid frogs reduced metabolic activities. The filaments with 20-40 nm width are connected to a ring component of the nuclear pore complex with 90-120 nm and extend up to 1æm (or more) into the nucleus. The filaments are curved and connect single or neighboring pores. The intranuclear filaments are associated with chromatin fibers and related to RNP particles of 20-25 nm and spheroidal structures of 0.5æm, with variations. The aggregates of several neighboring pores with the filaments are more commonly observed in 4n nuclei. We concluded that the intranuclear filaments may correspond to the fibrillar network described in Xenopus oocyte nucleus being probably related to RNA transport. The molecular basis of this RNA remains elusive. Nevertheless, the morphological aspects of the spheroidal structures indicate they could correspond to nucleolar chromatin or to nucleolus-derived structures. We also speculate whether the complex aggregates of neighboring pores with intranuclear filaments may correspond to pore clustering previously described in these tetraploid animals using freeze-etching experiments.

Filamentos ligados a poros foram visualizados em núcleos de espermatócitos de anuros através da técnica de espalhamento para microscopia eletrônica de transmissão. Os animais usados pertencem ao gênero Odontophrynus com espécies cripticas diplo e tetraplóides naturais, tendo os tetraplóides atividade metabólica reduzida. Os filamentos com 20-40 nm de largura são ligados a um anel componente do complexo poro nuclear de 90-120 nm e estendem-se até 1 æm (ou mais) para dentro do núcleo. Os filamentos são curvos e ligam poros simples ou poros vizinhos. Os filamentos intranucleares são associados a fibras de cromatina e relacionados a partículas de RNP de 20-25 nm e a estruturas esféricas de 0.5æm, com variações. Os agregados de poros vizinhos com os filamentos longos são mais freqüentemente observados em núcleos 4n. Concluímos que os filamentos intranucleares podem corresponder aos emaranhados fibrilares descritos em núcleos de oócitos de Xenopus e possivelmente relacion ados ao transporte de RNA. A base molecular desse RNA não é conhecida. Contudo, os aspectos morfológicos das estruturas esféricas parecem indicar que elas podem corresponder à cromatina nucleolar ou a estruturas derivadas do nucléolo. Também, especulamos se os agregados complexos de poros vizinhos com os filamentos intranucleares podem corresponder aos aglomerados de poros previamente descritos nesses animais tetraplóides através da técnica "freeze-etching".

Nerve cell nuclear and nucleolar abnormalities in the human oedematous cerebral cortex. An electron microscopic study using cortical biopsies.

Cerebral cortical biopsies of 17 patients with clinical diagnosis of congenital hydrocephalus, complicated brain trauma, cerebellar syndrome and vascular anomaly were examined with the transmission electron microscope to study the nuclear and nucleolar abnormalities induced by moderate and severe brain oedema, and the associated anoxic-ischemic conditions of brain tissue. In infant patients with congenital hydrocephalus and Arnold-Chiari malformation two different structural patterns of immature chromatin organization were found: the clear type characterized by a clear granular and fibrillar structure of euchromatin, scarce heterochromatin masses and few perichromatin granules, and a dense granular and fibrillar euchromatin with abundant and scattered heterochromatin masses, and increased number of perichromatin granules. The lobulated nuclei exhibited an irregularly dilated and fragmented perinuclear cistern, and areas of apparently intact nuclear pore complexes alternating with regions of nuclear pore complex disassembly. In moderate traumatic brain injuries some nucleoli exhibit apparent intact nucleolar substructures, and in severe brain oedema some nucleoli appeared shrunken and irregularly outlined with one or two fibrillar centers, and others were disintegrated. The nuclear and nucleolar morphological alterations are discussed in relation with oxidative stress, peroxidative damage, hemoglobin-induced cytotoxicity, calcium overload, glutamate excitotoxicity, and caspase activation.