Abstract. We have developed an in vitro system involving digitonin-permeabilized vertebrate cells to study biochemical events in the transport of macromolecules across the nuclear envelope. While treatment of cultured cells with digitonin permeabilizes the plasma membranes to macromolecules, the nuclear envelopes remain structurally intact and nuclei retain the ability to transport and accumulate proteins containing the SV40 large T antigen nuclear location sequence. Transport requires addition of exogenous cytosol to permeabilized cells, indicating the soluble cytoplasmic factor(s) required for nuclear import are released during digitonin treatment. In this reconstituted import system, a protein containing a nuclear location signal is rapidly accumulated in nuclei, where it reaches a 30-fold concentration compared to the surrounding medium within 30 min. Nuclear import is specific for a functional nuclear location sequence, requires ATP and cytosol, and is temperature dependent. Furthermore, accumulation of the transport substrate within nuclei is completly inhibited by wheat germ agglutinin, which binds to nuclear pore complexes and inhibits transport in vivo. Together, these results indicate that the permeabilized cell system reproduces authentic nuclear protein import. In a preliminary biochemical dissection of the system, we observe that the sulfhydryl alkylating reagent N-ethylmaleimide inactivates both cytosolic factor(s) and also component(s) in the insoluble permeabilized cell fraction required for nuclear protein import. Because this permeabilized cell model is simple, efficient, and works effectively with cells and cytosol fractions prepared from a variety of different vertebrate sources, it will prove powerful for investigating the biochemical pathway of nuclear transport.

The premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) is caused by a mutant lamin A (LA⌬50). Nuclei in cells expressing LA⌬50 are abnormally shaped and display a loss of heterochromatin. To determine the mechanisms responsible for the loss of heterochromatin, epigenetic marks regulating either facultative or constitutive heterochromatin were examined. In cells from a female HGPS patient, histone H3 trimethylated on lysine 27 (H3K27me3), a mark for facultative heterochromatin, is lost on the inactive X chromosome (Xi). The methyltransferase responsible for this mark, EZH2, is also down-regulated. These alterations are detectable before the changes in nuclear shape that are considered to be the pathological hallmarks of HGPS cells. The results also show a down-regulation of the pericentric constitutive heterochromatin mark, histone H3 trimethylated on lysine 9, and an altered association of this mark with heterochromatin protein 1␣ (Hp1␣) and the CREST antigen. This loss of constitutive heterochromatin is accompanied by an up-regulation of pericentric satellite III repeat transcripts. In contrast to these decreases in histone H3 methylation states, there is an increase in the trimethylation of histone H4K20, an epigenetic mark for constitutive heterochromatin. Expression of LA⌬50 in normal cells induces changes in histone methylation patterns similar to those seen in HGPS cells. The epigenetic changes described most likely represent molecular mechanisms responsible for the rapid progression of premature aging in HGPS patients.histone methylation ͉ heterochromatin ͉ progeria H utchinson-Gilford Progeria Syndrome (HGPS) is a premature aging disease usually diagnosed in the first 12-18 months of life (1). HGPS is characterized by a rapid progression of disorders including hair loss, growth retardation, lack of s.c. fat, aged-looking skin, osteoporosis, and arteriosclerosis (2, 3). Patients with HGPS usually die from heart attacks or strokes at Ϸ13 years (4). The common form of HGPS is caused by a conservative heterozygous mutation (1824 CϾT) in the human nuclear lamin A (LA) gene (LMNA), which introduces a splice site resulting in the synthesis of LA with 50 amino acids deleted near its C terminus [mutant LA (LA⌬50)] (5, 6).Nuclear lamins are divided into A and B types. Lamins A and C (LA͞C) are derived from LMNA by alternative splicing, whereas lamins B1 and B2 are derived from different genes. The B type lamins are expressed in every cell, whereas the expression of A type lamins is developmentally regulated. Lamins have a common tripartite structure with an ␣-helical central rod domain flanked by globular N-and C-terminal domains (7). The basic structural unit of lamins is a dimer consisting of two parallel and in-register protein chains that form a coiled coil through the association of their rod domains (8). Lamin dimers assemble in a head-to-tail fashion forming protofilaments that interact laterally to form numerous higher-order structures (9).Lamins are the major components of the nuclear lam...

The nuclear lamins function in the regulation of replication, transcription, and epigenetic modifications of chromatin. However, the mechanisms responsible for these lamin functions are poorly understood. We demonstrate that A-and B-type lamins form separate, but interacting, stable meshworks in the lamina and have different mobilities in the nucleoplasm as determined by fluorescence correlation spectroscopy (FCS). [Keywords: Lamins; chromatin; RNA polymerase II transcription; chromosome organization] Supplemental material is available at http://www.genesdev.org. Silencing lamin B1 (LB1) expression dramatically increases the lamina meshwork size and the mobility of nucleoplasmic lamin A (LA). The changes in lamina mesh

The cell nucleus must continually resist and respond to intercellular and intracellular mechanical forces to transduce mechanical signals and maintain proper genome organization and expression. Altered nuclear mechanics is associated with many human diseases, including heart disease, progeria, and cancer. Chromatin and nuclear envelope A-type lamin proteins are known to be key nuclear mechanical components perturbed in these diseases, but their distinct mechanical contributions are not known. Here we directly establish the separate roles of chromatin and lamin A/C and show that they determine two distinct mechanical regimes via micromanipulation of single isolated nuclei. Chromatin governs response to small extensions (<3 μm), and euchromatin/heterochromatin levels modulate the stiffness. In contrast, lamin A/C levels control nuclear strain stiffening at large extensions. These results can be understood through simulations of a polymeric shell and cross-linked polymer interior. Our results provide a framework for understanding the differential effects of chromatin and lamin A/C in cell nuclear mechanics and their alterations in disease.

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