Loss of lamin A function increases chromatin dynamics in the nuclear interior.

Chromatin is organized in a highly ordered yet dynamic manner in the cell nucleus, but the principles governing this organization remain unclear. Similarly, it is unknown whether, and how, various proteins regulate chromatin motion and as a result influence nuclear organization. Here by studying the dynamics of different genomic regions in the nucleus of live cells, we show that the genome has highly constrained dynamics. Interestingly, depletion of lamin A strikingly alters genome dynamics, inducing a dramatic transition from slow anomalous diffusion to fast and normal diffusion. In contrast, depletion of LAP2α, a protein that interacts with lamin A and chromatin, has no such effect on genome dynamics. We speculate that chromosomal inter-chain interactions formed by lamin A throughout the nucleus contribute to chromatin dynamics, and suggest that the molecular regulation of chromatin diffusion by lamin A in the nuclear interior is critical for the maintenance of genome organization.

Lamina-independent lamins in the nuclear interior serve important functions.

Nuclear lamins were originally described as the main constituents of the nuclear lamina, a filamentous meshwork closely associated with the inner nuclear membrane. However, within recent years, it has become increasingly evident that a fraction of lamins also resides throughout the nuclear interior. As intermediate-filament-type proteins, lamins have been suggested to fulfill mainly structural functions such as providing shape and mechanical stability to the nucleus. But recent findings show that both peripheral and nucleoplasmic lamins also have important roles in essential cellular processes such as transcription, DNA replication, cell cycle progression, and chromatin organization. Furthermore, more than 300 mutations in the gene encoding A-type lamins have been associated with several human diseases now generally termed laminopathies and comprising muscular dystrophies, lipodystrophies, cardiomyopathies, and premature aging diseases. This review focuses on the lamina-independent pool of lamins in the nuclear interior, which surprisingly has not been studied in much detail so far. We discuss the properties and regulation of nucleoplasmic lamins during the cell cycle, their interaction partners, and their potential involvement in cellular processes and the development of laminopathies.

The transcription factor ZEB1 (deltaEF1) promotes tumour cell dedifferentiation by repressing master regulators of epithelial polarity.

Epithelial to mesenchymal transition (EMT) is implicated in the progression of primary tumours towards metastasis and is likely caused by a pathological activation of transcription factors regulating EMT in embryonic development. To analyse EMT-causing pathways in tumourigenesis, we identified transcriptional targets of the E-cadherin repressor ZEB1 in invasive human cancer cells. We show that ZEB1 repressed multiple key determinants of epithelial differentiation and cell-cell adhesion, including the cell polarity genes Crumbs3, HUGL2 and Pals1-associated tight junction protein. ZEB1 associated with their endogenous promoters in vivo, and strongly repressed promotor activities in reporter assays. ZEB1 downregulation in undifferentiated cancer cells by RNA interference was sufficient to upregulate expression of these cell polarity genes on the RNA and protein level, to re-establish epithelial features and to impair cell motility in vitro. In human colorectal cancer, ZEB1 expression was limited to the tumour-host interface and was accompanied by loss of intercellular adhesion and tumour cell invasion. In invasive ductal and lobular breast cancer, upregulation of ZEB1 was stringently coupled to cancer cell dedifferentiation. Our data show that ZEB1 represents a key player in pathologic EMTs associated with tumour progression.

Intermediate filaments: novel assembly models and exciting new functions for nuclear lamins.

Intermediate filament (IF) proteins constitute a highly diverse family of fibrous proteins in metazoans, which assemble into 10-nm-thick filaments in the cytoplasm and the nucleus. Novel recent insights into the in vitro assembly mechanism have revealed principal differences in the formation of cytoplasmic and nuclear filaments. Moreover, the past years have seen dramatic developments for the nuclear specific IF proteins, the lamins. While in the past lamins have been assumed to form only a structural scaffold at the nuclear periphery, their discovery in the nuclear interior, the identification of novel lamin-binding proteins and the functional disruption of lamin structures have brought to light essential functions for lamins in fundamental cellular events such as chromatin organization, DNA replication and RNA transcription. Furthermore, mutations in lamins and lamin-binding proteins have been demonstrated to cause various different human diseases, affecting muscle, heart, neuronal, adipose and bone tissue or leading to premature ageing. However, the molecular basis of these diseases is just beginning to emerge.

E-cadherin regulates cell growth by modulating proliferation-dependent beta-catenin transcriptional activity.

beta-Catenin is essential for E-cadherin-mediated cell adhesion in epithelial cells, but it also forms nuclear complexes with high mobility group transcription factors. Using a mouse mammary epithelial cell system, we have shown previously that conversion of epithelial cells to a fibroblastoid phenotype (epithelial-mesenchymal transition) involves downregulation of E-cadherin and upregulation of beta-catenin transcriptional activity. Here, we demonstrate that transient expression of exogenous E-cadherin in both epithelial and fibroblastoid cells arrested cell growth or caused apoptosis, depending on the cellular E-cadherin levels. By expressing E-cadherin subdomains, we show that the growth-suppressive effect of E-cadherin required the presence of its cytoplasmic beta-catenin interaction domain and/or correlated strictly with the ability to negatively interfere with beta-catenin transcriptional activity. Furthermore, coexpression of beta-catenin or lymphoid enhancer binding factor-1 or T cell factor 3 with E-cadherin rescued beta-catenin transcriptional activity and counteracted E-cadherin-mediated cell cycle arrest. Stable expression of E-cadherin in fibroblastoid cells decreased beta-catenin activity and reduced cell growth. Since proliferating cells had a higher beta-catenin activity than G1 phase-arrested or contact-inhibited cells, we conclude that beta-catenin transcriptional activity is essential for cell proliferation and can be controlled by E-cadherin in a cell adhesion-independent manner.

Nuclear envelope and nuclear matrix: interactions and dynamics.

The peripheral nuclear lamina is located near the nuclear inner membrane and consists of lamin filaments and integral membrane proteins, including the lamin B receptor and various isoforms of lamina-associated polypeptides (LAP) 1 and 2. Several nuclear membrane proteins also interact with chromatin proteins BAF and Hp1. Lamins in the nuclear interior associate with at least one soluble (non-membrane-bound) LAP2 isoform named LAP2alpha. The internal lamins, together with Tpr-based filaments that connect to nuclear pore complexes, are proposed to be major structural elements of the internal nuclear matrix. We describe the structural links between the peripheral lamina and the internal nuclear matrix that are thought to be mediated by LAP2 family members, filament protein Tpr and nucleoporin Nup153. These findings are discussed in relation to human diseases that arise from mutations in nuclear lamina proteins.

Caspase-mediated cleavage of the chromosome-binding domain of lamina-associated polypeptide 2 alpha.

Lamina-associated polypeptide 2 alpha (LAP2 alpha) is a non-membrane-bound isoform of the LAP2 family involved in nuclear structure organization. Using various cell systems, including Jurkat, HL-60, and HeLa cells, and different death-inducing agents, such as anti-Fas antibody, topoisomerase inhibitors, and staurosporine, we found that LAP2 alpha was cleaved during apoptosis as rapidly as lamin B in a caspase-dependent manner yielding stable N- and C-terminal fragments of approximately 50 and 28 kDa, respectively. Based on fragment size and localization of immunoreactive epitopes, four potential cleavage sites were mapped between amino acids 403-485. These sites were located within a domain that has previously been described to be essential and sufficient for association of LAP2 alpha with chromosomes, suggesting that LAP2 alpha cleavage impairs its chromatin-binding properties. Immunofluorescence microscopy demonstrated that, unlike full length protein, apoptotic fragments did not colocalize with condensed chromatin, but remained in the nuclear compartment as long as a single nucleus was visible. Subfractionation analyses showed that the N-terminal LAP2 alpha fragment was extracted from intranuclear structures in detergent/salt buffers, whereas the C-terminal fragment remained associated with a residual framework devoid of chromatin. Our data suggest that early cleavage of LAP2 alpha) is important for chromatin reorganization during apoptosis.

Lamina-associated polypeptide 2alpha binds intranuclear A-type lamins.

The nucleoskeletal protein lamina-associated polypeptide 2(&agr;) (LAP2*) contains a large, unique C terminus and differs significantly from its alternatively spliced, mostly membrane-integrated isoforms, such as LAP2beta. Unlike lamin B-binding LAP2beta, LAP2alpha was found by confocal immunofluorescence microscopy to colocalize preferentially with A-type lamins in the newly formed nuclei assembled after mitosis. While only a subfraction of lamins A and C (lamin A/C) was associated with the predominantly nuclear LAP2alpha in telophase, the majority of lamin A/C colocalized with LAP2alpha in G(1)-phase nuclei. Furthermore, selective disruption of A-type lamin structures by overexpression of lamin mutants in HeLa cells caused a redistribution of LAP2alpha. Coimmunoprecipitation experiments revealed that a fraction of lamin A/C formed a stable, SDS-resistant complex with LAP2alpha in interphase cells and in postmetaphase cell extracts. Blot overlay binding studies revealed a direct binding of LAP2alpha to exclusively A-type lamins and located the interaction domains to the C-terminal 78 amino acids of LAP2alpha and to residues 319-566 in lamin A/C, which include the C terminus of the rod and the entire tail common to lamin A/C. These findings suggest that LAP2alpha and A-type lamins cooperate in the organization of internal nuclear structures.