Jean M. Underwood

Mitotic occupancy and lineage-specific transcriptional control of rRNA genes by Runx2

Regulation of ribosomal RNA genes is a fundamental process that supports the growth of cells and is tightly coupled with cell differentiation. Although rRNA transcriptional control by RNA polymerase I (Pol I) and associated factors is well studied, the lineage-specific mechanisms governing rRNA expression remain elusive. Runt-related transcription factors Runx1, Runx2 and Runx3 establish and maintain cell identity, and convey phenotypic information through successive cell divisions for regulatory events that determine cell cycle progression or exit in progeny cells. Here we establish that mammalian Runx2 not only controls lineage commitment and cell proliferation by regulating genes transcribed by RNA Pol II, but also acts as a repressor of RNA Pol I mediated rRNA synthesis. Within the condensed mitotic chromosomes we find that Runx2 is retained in large discrete foci at nucleolar organizing regions where rRNA genes reside. These Runx2 chromosomal foci are associated with open chromatin, co-localize with the RNA Pol I transcription factor UBF1, and undergo transition into nucleoli at sites of rRNA synthesis during interphase. Ribosomal RNA transcription and protein synthesis are enhanced by Runx2 deficiency that results from gene ablation or RNA interference, whereas induction of Runx2 specifically and directly represses rDNA promoter activity. Runx2 forms complexes containing the RNA Pol I transcription factors UBF1 and SL1, co-occupies the rRNA gene promoter with these factors in vivo, and affects local chromatin histone modifications at rDNA regulatory regions. Thus Runx2 is a critical mechanistic link between cell fate, proliferation and growth control. Our results suggest that lineage-specific control of ribosomal biogenesis may be a fundamental function of transcription factors that govern cell fate.

Pulse energy dependence of subcellular dissection by femtosecond laser pulses

Precise dissection of cells with ultrashort laser pulses requires a clear understanding of how the onset and extent of ablation (i.e., the removal of material) depends on pulse energy. We carried out a systematic study of the energy dependence of the plasma-mediated ablation of fluorescently-labeled subcellular structures in the cytoskeleton and nuclei of fixed endothelial cells using femtosecond, near-infrared laser pulses focused through a high-numerical aperture objective lens (1.4 NA). We find that the energy threshold for photobleaching lies between 0.9 and 1.7 nJ. By comparing the changes in fluorescence with the actual material loss determined by electron microscopy, we find that the threshold for true material ablation is about 20% higher than the photobleaching threshold. This information makes it possible to use the fluorescence to determine the onset of true material ablation without resorting to electron microscopy. We confirm the precision of this technique by severing a single microtubule without disrupting the neighboring microtubules, less than 1 micrometer away.

Ectopic Runx2 Expression in Mammary Epithelial Cells Disrupts Formation of Normal Acini Structure: Implications for Breast Cancer Progression

The transcription factor Runx2 is highly expressed in breast cancer cells compared with mammary epithelial cells and contributes to metastasis. Here we directly show that Runx2 expression promotes a tumor cell phenotype of mammary acini in three-dimensional culture. Human mammary epithelial cells (MCF-10A) form polarized, growth-arrested, acini-like structures with glandular architecture. The ectopic expression of Runx2 disrupts acini formation, and electron microscopic ultrastructural analysis revealed the absence of lumens. Characterization of the disrupted acini structures showed increased cell proliferation (Ki-67 positive cells), decreased apoptosis (Bcl-2 induction), and loss of basement membrane formation (absence of beta(4) integrin expression). In complementary experiments, inhibition of Runx2 function in metastatic MDA-MB-231 breast cancer cells by stable expression of either short hairpin RNA-Runx2 or a mutant Runx2 deficient in subnuclear targeting resulted in reversion of acini to more normal structures and reduced tumor growth in vivo. These novel findings provide direct mechanistic evidence for the biological activity of Runx2, dependent on its subnuclear localization, in promoting early events of breast cancer progression and suggest a molecular therapeutic target.

The Ultrastructure of MCF-10A Acini

MCF‐10A human mammary epithelial cells cultured inside reconstituted basement membrane form acini that resemble the acinar structures of mammary lobules. This three‐dimensional culture system has been used for identifying and characterizing the signal transduction pathways controlling cell proliferation and death, and for studying their disregulation in malignant progression. We have compared the ultrastructure of MCF‐10A acini, MCF‐10A cells grown in monolayer, and the acinar structures of human breast lobules. The tissue architecture of MCF‐10A acini was formed by hemidesmosomes connected to a basement membrane and by abundant desmosomes between acinar cells. Intermediate filaments that joined into large and abundant filament bundles connected hemidesmosomes and desmosomes to sites at the nuclear surface. Fewer and thinner bundles of filaments were observed in monolayer MCF‐10A cells and even fewer in breast tissue. Tight junctions were observed between cells in breast tissue but missing in MCF‐10A acini. The cytoplasm of MCF‐10A acinar cells had a polar organization similar to that observed in breast tissue, with centrosomes and the Golgi apparatus on the apical side of the nucleus. MCF‐10A acinar nuclei had an irregular, frequently invaginated surface and had a single nucleolus. The distribution of heterochromatin was similar to that in the epithelial cells of breast tissue. The nuclei of monolayer MCF‐10A cells had multiple nucleoli, a more regular profile, and less heterochromatin. Electron microscopy has the resolution required to survey features of MCF‐10A cell and acinus architecture that may change with manipulations designed to induce malignant phenotypes. J. Cell. Physiol.

Binding of ATP to UAP56 is necessary for mRNA export

The major-histocompatibility-complex protein UAP56 (BAT1) is a DEAD-box helicase that is deposited on mRNA during splicing. UAP56 is retained on spliced mRNA in an exon junction complex (EJC) or, alternatively, with the TREX complex at the 5′ end, where it might facilitate the export of the spliced mRNA to the cytoplasm. Using confocal microscopy, UAP56 was found to be concentrated in RNA-splicing speckled domains of nuclei but was also enriched in adjacent nuclear regions, sites at which most mRNA transcription and splicing occur. At speckled domains, UAP56 was in complexes with the RNA-splicing and -export protein SRm160, and, as measured by FRAP, was in a dynamic binding equilibrium. The application of an in vitro FRAP assay, in which fluorescent nuclear proteins are photobleached in digitonin-extracted cells, revealed that the equilibrium binding of UAP56 in complexes at speckled domains was directly regulated by ATP binding. This was confirmed using a point mutant of UAP56 that did not bind ATP. Point mutation of UAP56 to eliminate ATP binding did not affect RNA splicing, but strongly inhibited the export of mRNA to the cytoplasm.

Endothelium and “silver lines”

The significance of endothelial “silver lines” was studied by TEM in rat aortas after perfusion with glutaraldehyde followed by silver nitrate. Standard TEM technique proved unsatisfactory (coarse silver granules, imprecise localization, artefacts). Exposure of the silver-treated aortas to photographic fixer markedly improved the image of the deposits leaving fine, stable, uniform “residual granules” about 100 Å in diameter. Most of these granules were localized along the intercellular junctions; they also tended to pool in the basement membrane beneath each junction. This image suggests that the Ag+ ions pass through the junction, and react with its contents as well as with the basement membrane beyond it. A scheme is proposed to explain the reaction of Ag+ ions with anions and negatively charged radicals within the junction. It is concluded that the “silver lines” represent not only a histochemical effect, but also the visualization of a transendothelial electrolyte pathway.

Nuclear shape changes are induced by knockdown of the SWI/SNF ATPase BRG1 and are independent of cytoskeletal connections.

Changes in nuclear morphology occur during normal development and have been observed during the progression of several diseases. The shape of a nucleus is governed by the balance of forces exerted by nuclear-cytoskeletal contacts and internal forces created by the structure of the chromatin and nuclear envelope. However, factors that regulate the balance of these forces and determine nuclear shape are poorly understood. The SWI/SNF chromatin remodeling enzyme ATPase, BRG1, has been shown to contribute to the regulation of overall cell size and shape. Here we document that immortalized mammary epithelial cells show BRG1-dependent nuclear shape changes. Specifically, knockdown of BRG1 induced grooves in the nuclear periphery that could be documented by cytological and ultrastructural methods. To test the hypothesis that the observed changes in nuclear morphology resulted from altered tension exerted by the cytoskeleton, we disrupted the major cytoskeletal networks and quantified the frequency of BRG1-dependent changes in nuclear morphology. The results demonstrated that disruption of cytoskeletal networks did not change the frequency of BRG1-induced nuclear shape changes. These findings suggest that BRG1 mediates control of nuclear shape by internal nuclear mechanisms that likely control chromatin dynamics.

β1 integrins mediate cell proliferation in three-dimensional cultures by regulating expression of the sonic hedgehog effector protein, GLI1

The β1 integrins play an important role in the modulation of cancer cell proliferation and tumor growth. We have previously shown that β1 integrins associate with insulin‐like growth factor type 1 receptor (IGF‐IR) and regulate IGF‐stimulated prostate cancer cell proliferation. In the present study, we find that downregulation of β1 in prostate cancer cells inhibits IGF‐IR and AKT activation. We also show that β1 downregulation in two‐ and three‐dimensional (3D) prostate cancer cell cultures significantly reduces expression of GLI1, a transcription factor known to be regulated by the IGF/AKT signaling pathway and to be a downstream effector of sonic hedgehog. Re‐expression of GLI1 rescues the inhibitory effect of β1 downregulation on prostate cancer cell proliferation in 3D cultures. We find that downregulation of β1 significantly reduces surface expression of the associated α5 integrin subunit. Our results indicate that the β1/IGF–IR complex regulates expression of GLI1, which in turn promotes cancer cell proliferation in 3D cultures. J. Cell. Physiol. 224:210–217, 2010

The significance of endothelial stomata and stigmata in the rat aorta: An electron microscopic study

Perfusion of arteries with dilute silver nitrate produces in the endothelium (a) a pattern of pericellular black lines, which we earlier interpreted as a marker of the physiological electrolyte pathway (Zand et al. 1982), and (b) focal black deposits on or between the cells, either ring-shaped (stomata) or solid (stigmata). The purpose of this study was to clarify the nature and significance of these controversial structures. A glutaraldehyde-fixednormal rat aorta was perfused with silver nitrate; 17 typical stomata and stigmata were photographeden face, then studied on ultrathin serial sections. When seenen face, they fell into three groups: (I) 4 stomata in endothelial cells; (II) 6 stigmata in endothelial cells; (III) 7 stigmata on intercellular junctions. By electron microscopy, (I) all thestomata in endothelial cells corresponded to myoendothelial herniae. (II) Of the 6stigmata in endothelial cells, 4 corresponded again to myoendothelial herniae, 2 corresponded to blebs (it seemed likely that these blebs had existedin vivo, but the possibility of a fixation artefact could not be excluded). (III) Of the 7stigmata on intercellular junctions, one corresponded to the diapedesis of a mononuclear cell; the other 6 did not correspond to visible endothelial changes and are best interpreted as points of normally higher permeability. We conclude that stomata and stigmata (under the conditions of our experiments) can be explained in at least 4 different ways, depending in part on their location (in cells, on junctions). These ancient terms therefore remain useful for descriptive purposes, as long as it is realized that their significance in any given case must be determined by electron microscopic study.

Endothelial Permeability in Inflammation

The electron microscopic studies of N. Simionescu and collaborators17–19 have clearly shown that the endothelium in the three basic segments of the microcirculation—arterioles, capillaries, and venules—differs in terms of ultrastructure and permeability. Segmental differences are obvious also in the inflammatory response. Indeed, those chemical mediators that increase microvascular permeability do so by affecting selectively the permeability of the venules; this was established by using a method that selectively marks leaky segments of the microcirculation, namely vascular labeling.13 A clue to this selectivity for the venules was provided by Heltianu et al. 5 who showed that the endothelium of these vessels is especially rich in histamine receptors. Another difference between capillary and venular endothelium is that the latter is able to contract in vivo under the stimulus of inflammatory mediators7; rather than reducing the caliber of the vessel, this contraction produces gaps between the endothelial cells, allowing the escape of fluid.7,14