Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus

Abstract

What determines the nuclear organization within a cell and whether this organization itself can impose cellular function within a tissue remains unknown. To explore the relationship between nuclear organization and tissue architecture and function, we used a model of human mammary epithelial cell acinar morphogenesis. When cultured within a reconstituted basement membrane (rBM), HMT-3522 cells form polarized and growth-arrested tissue-like acini with a central lumen and deposit an endogenous BM. We show that rBM-induced morphogenesis is accompanied by relocalization of the nuclear matrix proteins NuMA, splicing factor SRm160, and cell cycle regulator Rb. These proteins had distinct distribution patterns specific for proliferation, growth arrest, and acini formation, whereas the distribution of the nuclear lamina protein, lamin B, remained unchanged. NuMA relocalized to foci, which coalesced into larger assemblies as morphogenesis progressed. Perturbation of histone acetylation in the acini by trichostatin A treatment altered chromatin structure, disrupted NuMA foci, and induced cell proliferation. Moreover, treatment of transiently permeabilized acini with a NuMA antibody led to the disruption of NuMA foci, alteration of histone acetylation, activation of metalloproteases, and breakdown of the endogenous BM. These results experimentally demonstrate a dynamic interaction between the extracellular matrix, nuclear organization, and tissue phenotype. They further show that rather than passively ref lecting changes in gene expression, nuclear organization itself can modulate the cellular and tissue phenotype.The cell nucleus is organized by a nonchromatin internal structure referred to as the nuclear matrix (NM; refs. 1-3). Identified NM components include coiled-coil proteins (4), cell cycle regulators (5), tissue-specific transcription factors (6, 7), and RNA splicing factors (for review see ref.2). Although splicing factors have been shown to redistribute during cellular differentiation (8, 9) and following the induction of gene expression (10), spatial distribution of nuclear components are thought to be the consequence of changes in gene expression (8, 10, 11). However, whether NM composition and structure may themselves affect gene expression and cellular function has not been examined.To systematically study the effect of cell growth and tissue differentiation on nuclear organization, we used a reconstituted basement membrane (rBM)-directed model of mammary gland morphogenesis (12). The HMT-3522 human mammary epithelial cells (HMECs) were isolated from reduction mammoplasty and became immortalized in culture (13). When embedded within a rBM, these cells arrest growth, organize an endogenous BM, and form polarized acinus-like structures with vectorial secretion of sialomucin into a central lumen (12). We used this model to compare the nuclear organization of HMECs cultured on a plastic surface [two-dimensional (2D) monolayer] vs. a three-dimensional (3D) rBM. Nuclear organization was assessed by examini...

NuMA Influences Higher Order Chromatin Organization in Human Mammary Epithelium

“…To verify that no cross-contamination between fractions had occurred during preparation and that NuMA was indeed behaving similarly to chromatin-associated proteins and differently from nuclear matrix proteins, the same nitrocellulose membranes were blotted for chromatin-associated protein MCM3 (Takei et al, 2001) and nuclear matrix protein lamin B. MCM3 was enriched in the chromatin compartment (ϳ81% of the total amount proceeding from the chromatin and nondigestible nuclear fractions, as measured by densitometry) and lamin B was enriched in the nondigestible nuclear fraction (ϳ93% of the total amount proceeding from the chromatin and nondigestible nuclear fractions, as measured by densitometry), indicating that the fractionation had been successful ( Figure 2B). A change in the distribution of NuMA upon breast acinar differentiation is shown by the formation of distinct and large foci ( Figure 1D; Lelièvre et al, 1998). This process is recapitulated upon culture of nonneoplastic S1 HMECs under 3D conditions in the presence of Matrigel.…”

“…When antibodies directed against the C terminus of NuMA are introduced into mammary acini in 3D culture, the distinct NuMA foci that characterize differentiated mammary epithelial cells are replaced rapidly by the diffuse pattern characteristic of nondifferentiated cells. This redistribution is accompanied by loss of acinar differentiation, as shown by degradation of the basement membrane and alteration of acinar polarity (Lelièvre et al, 1998). Thus, a large body of data about NuMA in the interphase nucleus points to a role for this protein in differentiation and suggests a relationship between NuMA and chromatin.…”

“…Subsequently NuMA, variously named SPN antigen, p240 antigen, centrophilin, 1F1/1H1 antigen, SP-H antigen, and W1 antigen (Compton et al, 1991(Compton et al, , 1992Kallajoki et al, 1991Kallajoki et al, , 1993Maekawa et al, 1991;Tousson et al, 1991;Yang et al, 1992;Maekawa and Kuriyama, 1993;Tang et al, 1993), was shown to be a critical player in the formation and stabilization of the mitotic spindle, notably by associating with the minus end of spindle microtubules and interacting with dynein, dynactin, and LGN (Compton and Cleveland, 1993;Gaglio et al, 1995;Merdes et al, 2000;Du et al, 2001;Gehmlich et al, 2004). In addition to its location at the poles of the mitotic spindle, NuMA has been reported in the nucleus of both cycling and growth-arrested cells, as shown by immunostaining of cells in culture and tissue biopsy sections (Lelièvre et al, 1998;Merdes and Cleveland 1998;Gribbon et al, 2002;Taimen et al, 2004). However, a role for NuMA in interphase remains to be determined.…”

“…© 2007 by The American Society for Cell Biology glandular structures (acini) upon three-dimensional (3D) culture in the presence of laminin-rich Matrigel (Lelièvre et al, 1998). During differentiation of lens epithelial cells, NuMA colocalizes with the Cajal bodies and with nuclear speckles that contain components of the spliceosome (Gribbon et al, 2002).…”

“…We have shown that introduction of antibodies directed against the C terminus of NuMA in acini modifies the nuclear distribution of NuMA and induces the loss of acinar differentiation by altering basal polarity, as evidenced by the lack of continuous staining for collagen IV, a major component of the basement membrane (Lelièvre et al, 1998). Interestingly, the alteration of NuMA organization was accompanied by changes in the distribution of the chromatin marker acetyl-H4 (Lelièvre et al, 1998), suggesting that altering NuMA function at its C terminus may affect chromatin organization. NuMA-CT spans amino acids 1701-2101 and the distal portion (starting at amino acid 1915) of the C terminus of NuMA (NuMA-CTDP) is highly conserved in vertebrates (Abad et al, 2004), suggesting a potentially important role for this region in NuMA function.…”