Microtubule-organizing center-mediated structural atypia in low- and high-grade urothelial carcinoma.

Urothelial carcinoma (UC) comprises two subtypes, low grade (LG-UC) and high grade (HG-UC), with different pathological and clinical behavior. LG-UC and HG-UC are classified based on cellular and structural atypia of pathological findings. The mechanisms responsible for maintaining structural atypia, such as the disturbance of nuclear polarity, remain unclear. In this study, we studied microtubule-organizing center (MTOC)-mediated nuclear polarity in UC subtypes. We evaluated six cases with normal urothelium (NU), 10 LG-UC cases, and 10 HG-UC cases by double immunofluorescence staining of γ-tubulin as a marker of MTOC and E-cadherin as a marker of each cell border. The number and position of γ-tubulin dots of expression in more than 100 cells per case were assessed using the spatial relationship with the nucleus and surface-basal axis. We found one γ-tubulin dot in most normal and tumor cells, and more than two γ-tubulin dots in 4.6% of NU cells, 6.1% of LG-UC cells, and 9.8% of HG-UC cells. More than three γ-tubulin dots were found only in 1.2% of HG-UC cells. Surface side positioning of γ-tubulin was found in 77.4% of normal urothelial cells, 63.8% of LG-UC cells, and 39.2% of HG-UC cells, whereas aberrant lateral and basal side positioning of γ-tubulin was found in 22.6% of normal urothelial cells, 36.1% of LG-UC cells, and 60.8% of HG-UC cells. We concluded that numerical and positional aberrations of MTOC in UC cases were strongly correlated with both cellular and structural atypia as well as abnormal cell proliferation.

The centrosomal/basal body protein OFD1 is required for microtubule organization and cell cycle progression.

Oral-Facial-Digital type I (OFD1) is a rare inherited form of renal cystic disease associated with ciliary dysfunction. This disorder is due to mutations in the OFD1 gene that encodes a protein localized to centrosomes and basal bodies in different cell types. Immunofluorescence analysis demonstrated that OFD1 displays a dynamic distribution during cell cycle. High-content microscopy analysis of Ofd1-depleted fibroblasts revealed impaired cell cycle progression. Immunofluorescence analysis and cell proliferation assays also indicated the presence of a variety of defects such as centrosome accumulation, nuclear abnormalities and aneuploidy. In addition, Ofd1-depleted cells displayed an abnormal microtubule network that may underlie these defects. All together our results suggest that OFD1 contributes to the function of the microtubule organizing center (MTOC) in the cell, controlling cell cycle progression both in vitro and in vivo.

Spectrin alpha is important for rear polarization of the microtubule organizing center during migration and spindle pole assembly during division of neointimal smooth muscle cells.

Directed migration of smooth muscle cells (SMCs) from the media to the intima and their subsequent proliferation are key events in atherosclerosis as these cells contribute to the bulk and stability of atheromatous plaques. We showed previously that two cytoskeleton-associated proteins, RHAMM and ARPC5, play important roles in rear polarization of the microtubule organizing centre (MTOC), directed migration, and in maintaining cell division fidelity. These proteins were analyzed to predict additional potential interacting partners using the bioinformatics programs BLAST, ClustalW, and PPI Spider. We identified spectrin alpha, a protein with a known role in actin polymerization as part of the pathway. We show that in migrating SMCs spectrin alpha localizes at the nodes of the actin net, and it partially colocalizes with RHAMM in the perinuclear region. In dividing SMCs spectrin alpha is present at spindle poles and midbody. Moreover, we show that spectrin alpha and RHAMM interact in a complex. Using siRNA to knockdown spectrin disrupted SMC migration, MTOC polarization, and the assembly of a polygonal actin net dorsolateral of the nucleus. Spectrin alpha knockdown also disrupted the organization of the bipolar spindle, chromosome division, and cytokinesis during cell division. The identification of interacting partners such as spectrin alpha and the decoding of pathways involved in polarity regulation during the migration of smooth muscle cells in atherosclerosis is important for identifying atherosclerosis biomarkers and developing therapeutic agents to block atherosclerotic plaque formation.

Microtubule-organizing center-mediated nuclear polarity in various normal and neoplastic human tissues.

Nuclear polarity is characterized by intracytoplasmic nuclear positioning and alignment in the tissue. The mechanisms responsible for maintaining nuclear polarity in normal cells and its disturbance in neoplastic cells are not understood. We studied microtubule-organizing center (MTOC) positioning-mediated nuclear polarity in various normal and neoplastic human tissues, as well as in cultured cells. To visualize the MTOC in cells, gamma-tubulin and pericentrin were immunohistochemically stained by fluorescence and non-fluorescence methods. Position of MTOC in normal and neoplastic tissue was assessed by spatial relationship with nucleus and apico-basal axis. We found MTOC positioning to be related to morphogenesis in various normal and neoplastic human tissues, as well as in cultured cells. MTOC positions were different between two-dimensional cultured isolated cells and three-dimensional cultured gland-formed cells. The MTOC position was specific depending on the cell type in the tissue structure. In particular, glandular and urothelial epithelium had a strong relationship with preservation of nuclear polarity and MTOC positioning. Carcinoma cells showed an irregular position or absence of the MTOC depending on poorer differentiation and higher grade of carcinomas. In conclusion, the position of the MTOC affects regulation of nuclear polarity and morphogenesis of normal and pathological tissue structure.

ACF7: an essential integrator of microtubule dynamics.

ACF7 is a member of the spectraplakin family of cytoskeletal crosslinking proteins possessing actin and microtubule binding domains. Here, we show that ACF7 is an essential integrator of MT-actin dynamics. In endodermal cells, ACF7 binds along microtubules but concentrates at their distal ends and at cell borders when polarized. In ACF7's absence, microtubules still bind EB1 and CLIP170, but they no longer grow along polarized actin bundles, nor do they pause and tether to actin-rich cortical sites. The consequences are less stable, long microtubules with skewed cytoplasmic trajectories and altered dynamic instability. In response to wounding, ACF7 null cultures activate polarizing signals, but fail to maintain them and coordinate migration. Rescue of these defects requires ACF7's actin and microtubule binding domains. Thus, spectraplakins are important for controlling microtubule dynamics and reinforcing links between microtubules and polarized F-actin, so that cellular polarization and coordinated cell movements can be sustained.

Deletion of exon 4 from human surfactant protein C results in aggresome formation and generation of a dominant negative.

Human surfactant protein C (hSP-C) is synthesized by the alveolar type 2 cell as a 197 amino acid integral membrane proprotein and proteolytically processed to a secreted 3.7 kDa mature form. Although the SP-C null mouse possesses a non-lethal phenotype, a heterozygous substitution of A for G in the first base of intron 4 of the human SP-C gene (c.460+1A>G) has been reported in association with familial interstitial lung disease and absence of mature protein. This mutation produces a splice deletion of exon 4 (deltaExon4) resulting in removal of a positionally conserved cysteine in the C-terminal flanking propeptide. Based on a prior study showing that an identical deletion in the rat isoform diverted mutant protein to stable aggregates, we hypothesized that expression of the deltaExon4 mutation would result in disruption of intracellular trafficking of both mutant and wild-type proSP-C. We tested this in vitro using fusion proteins of EGFP conjugated either to wild-type SP-C (EGFP/hSP-C(1-197)) or to SP-C deleted of Exon4 (EGFP/hSP-C(deltaExon4)). Fluorescence microscopy showed that EGFP/hSP-C(1-197) transfected into A549 cells was expressed in a punctuate pattern in CD63 (+) cytoplasmic vesicles, whereas EGFP/hSP-C(deltaExon4) accumulated in ubiquitinated perinuclear inclusions linked to the microtubule organizing center. A similar juxtanuclear pattern was observed following transfection of SP-C cDNA lacking only cysteine residues in the C-terminal propeptide encoded by Exon 4 (EGFP/hSP-C(C120/121G)). To evaluate whether mutant proSP-C could function as a dominant negative, EGFP/hSP-C(deltaExon4) was cotransfected with HA-tagged hSP-C(1-197) and resulted in the restriction of both forms to perinuclear compartments. Addition of Na(+) 4-phenylbutyrate, a facilitator of trafficking of other misfolded proteins, attenuated the aggregation of EGFP/hSP-C(deltaExon4). We conclude that c.460+1A>G mutation of human SP-C results in disruption of disulfide-mediated folding encoded by Exon 4 leading to diversion of unprocessed proSP-C to aggresomes. The heterotypic oligomerization of hSP-C(1-197) and hSP-C(deltaExon4) provides a molecular mechanism for the dominant-negative effect observed in vivo.

Expanded polyglutamine stretches form an 'aggresome'.

To understand the pathogenetic mechanisms underlying polyglutamine (polyQ) diseases, we investigated the mechanisms of the formation of aggregate bodies containing expanded polyQ stretches, focusing on dentatorubral-pallidoluysian atrophy (DRPLA). We demonstrated that the expression of a truncated DRPLA protein containing expanded polyQ stretches in COS-7 cells resulted in the formation of perinuclear aggregate bodies that are co-localized with gamma-tubulin, a protein marker for the microtubules-organizing center (MTOC). A collapsed vimentin network surrounded these aggregate bodies. Furthermore, disruption of the microtubules (MTs) with nocodazole resulted in the formation of small aggregate bodies that were scattered throughout the cytoplasm. These findings suggest that the truncated DRPLA proteins containing expanded polyQ stretches unfold and form small aggregate bodies in the cell periphery. These aggregates move on MTs to the MTOC, where they remain as distinct 'aggresomes''.