Quantitative high throughput screening using a primary human three-dimensional organotypic culture predicts in vivo efficacy.

The tumour microenvironment contributes to cancer metastasis and drug resistance. However, most high throughput screening (HTS) assays for drug discovery use cancer cells grown in monolayers. Here we show that a multilayered culture containing primary human fibroblasts, mesothelial cells and extracellular matrix can be adapted into a reliable 384- and 1,536-multi-well HTS assay that reproduces the human ovarian cancer (OvCa) metastatic microenvironment. We validate the identified inhibitors in secondary in vitro and in vivo biological assays using three OvCa cell lines: HeyA8, SKOV3ip1 and Tyk-nu. The active compounds directly inhibit at least two of the three OvCa functions: adhesion, invasion and growth. In vivo, these compounds prevent OvCa adhesion, invasion and metastasis, and improve survival in mouse models. Collectively, these data indicate that a complex three-dimensional culture of the tumour microenvironment can be adapted for quantitative HTS and may improve the disease relevance of assays used for drug screening.

Mesothelial cells promote early ovarian cancer metastasis through fibronectin secretion.

Ovarian cancer (OvCa) metastasizes to organs in the abdominal cavity, such as the omentum, which are covered by a single layer of mesothelial cells. Mesothelial cells are generally thought to be "bystanders" to the metastatic process and simply displaced by OvCa cells to access the submesothelial extracellular matrix. Here, using organotypic 3D cultures, we found that primary human mesothelial cells secrete fibronectin in the presence of OvCa cells. Moreover, we evaluated the tumor stroma of 108 human omental metastases and determined that fibronectin was consistently overexpressed in these patients. Blocking fibronectin production in primary mesothelial cells in vitro or in murine models, either genetically (fibronectin 1 floxed mouse model) or via siRNA, decreased adhesion, invasion, proliferation, and metastasis of OvCa cells. Using a coculture model, we determined that OvCa cells secrete TGF-ß1, which in turn activates a TGF-ß receptor/RAC1/SMAD-dependent signaling pathway in the mesothelial cells that promotes a mesenchymal phenotype and transcriptional upregulation of fibronectin. Additionally, blocking α5 or ß1 integrin function with antibodies reduced metastasis in an orthotopic preclinical model of OvCa metastasis. These findings indicate that cancer-associated mesothelial cells promote colonization during the initial steps of OvCa metastasis and suggest that mesothelial cells actively contribute to metastasis.

Three-dimensional modeling of ovarian cancer.

New models for epithelial ovarian cancer initiation and metastasis are required to obtain a mechanistic understanding of the disease and to develop new therapeutics. Modeling ovarian cancer however is challenging as a result of the genetic heterogeneity of the malignancy, the diverse pathology, the limited availability of human tissue for research, the atypical mechanisms of metastasis, and because the origin is unclear. Insights into the origin of high-grade serous ovarian carcinomas and mechanisms of metastasis have resulted in the generation of novel three-dimensional (3D) culture models that better approximate the behavior of the tumor cells in vivo than prior two-dimensional models. The 3D models aim to recapitulate the tumor microenvironment, which has a critical role in the pathogenesis of ovarian cancer. Ultimately, findings using models that accurately reflect human ovarian cancer biology are likely to translate into improved clinical outcomes. In this review we discuss the design of new 3D culture models of ovarian cancer primarily using human cells, key studies in which these models have been applied, current limitations, and future applications.

Binding of the CYK-4 subunit of the centralspindlin complex induces a large scale conformational change in the kinesin subunit.

Centralspindlin is a critical regulator of cytokinesis in animal cells. It is a tetramer consisting of ZEN-4/MKLP1, a kinesin-6 motor, and CYK-4/MgcRacGAP, a Rho GTPase-activating protein. At anaphase, centralspindlin localizes to a narrow region of antiparallel microtubule overlap and initiates central spindle assembly. Central spindle assembly requires complex formation between ZEN-4 and CYK-4. However, the structural consequences of CYK-4 binding to ZEN-4 are unclear as are the mechanisms of microtubule bundling. Here we investigate whether CYK-4 binding induces a conformational change in ZEN-4. Characterization of the structure and conformational dynamics of the minimal interacting regions between ZEN-4 and CYK-4 by continuous wave EPR and double electron-electron resonance (DEER) spectroscopy reveals that CYK-4 binding dramatically stabilizes the relative positions of the neck linker regions of ZEN-4. Additionally, our data indicate that each neck linker is similarly structured in the bound and unbound states. CYK-4 binding decreases the rate of ZEN-4-mediated microtubule gliding. These results constrain models for the molecular organization of centralspindlin.

Centralspindlin: at the heart of cytokinesis.

The final step in the cell cycle is the formation of two genetically identical daughter cells by cytokinesis. At the heart of cytokinesis in animal cells is the centralspindlin complex which is composed of two proteins, a kinesin-like protein, Mitotic kinesin-like protein 1, and a Rho GTPase activating protein (RhoGAP), CYK-4. Through its targeted localization to a narrow region of antiparallel microtubule overlap immediately following chromosome segregation, centralspindlin initiates central spindle assembly. Centralspindlin has several critical functions during cell division including positioning of the division plane, regulation of Rho family GTPases, as well as midbody assembly and abscission. In this review, we will examine the biochemistry of centralspindlin and its multiple functions during cell division. Remarkably, several of its critical functions are somewhat unexpected. Although endowed with motor domains, centralspindlin has an important role in generating stable, antiparallel microtubule bundles. Although it contains a Rho family GAP domain, it has a central role in the activation of RhoA during cytokinesis. Finally, centralspindlin functions as a motor protein complex, as a scaffold protein for key regulators of abscission and as a conventional RhoGAP. Because of these diverse functions, centralspindlin lies at the heart of the cytokinetic mechanism.

Mps1 phosphorylation of Dam1 couples kinetochores to microtubule plus ends at metaphase.

BACKGROUND: Duplicated chromosomes are equally segregated to daughter cells by a bipolar mitotic spindle during cell division. By metaphase, sister chromatids are coupled to microtubule (MT) plus ends from opposite poles of the bipolar spindle via kinetochores. Here we describe a phosphorylation event that promotes the coupling of kinetochores to microtubule plus ends. RESULTS: Dam1 is a kinetochore component that directly binds to microtubules. We identified DAM1-765, a dominant allele of DAM1, in a genetic screen for mutations that increase stress on the spindle pole body (SPB) in Saccharomyces cerevisiae. DAM1-765 contains the single mutation S221F. We show that S221 is one of six Dam1 serines (S13, S49, S217, S218, S221, and S232) phosphorylated by Mps1 in vitro. In cells with single mutations S221F, S218A, or S221A, kinetochores in the metaphase spindle form tight clusters that are closer to the SPBs than in a wild-type cell. Five lines of experimental evidence, including localization of spindle components by fluorescence microscopy, measurement of microtubule dynamics by fluorescence redistribution after photobleaching, and reconstructions of three-dimensional structure by electron tomography, combined with computational modeling of microtubule behavior strongly indicate that, unlike wild-type kinetochores, Dam1-765 kinetochores do not colocalize with an equal number of plus ends. Despite the uncoupling of the kinetochores from the plus ends of MTs, the DAM1-765 cells are viable, complete the cell cycle with the same kinetics as wild-type cells, and biorient their chromosomes as efficiently as wild-type cells. CONCLUSIONS: We conclude that phosphorylation of Dam1 residues S218 and S221 by Mps1 is required for efficient coupling of kinetochores to MT plus ends. We find that efficient plus-end coupling is not required for (1) maintenance of chromosome biorientation, (2) maintenance of tension between sister kinetochores, or (3) chromosome segregation.

Basal body duplication and maintenance require one member of the Tetrahymena thermophila centrin gene family.

Centrins, small calcium binding EF-hand proteins, function in the duplication of a variety of microtubule organizing centers. These include centrioles in humans, basal bodies in green algae, and spindle pole bodies in yeast. The ciliate Tetrahymena thermophila contains at least four centrin genes as determined by sequence homology, and these have distinct localization and expression patterns. CEN1's role at the basal body was examined more closely. The Cen1 protein localizes primarily to two locations: one is the site at the base of the basal body where duplication is initiated. The other is the transition zone between the basal body and axoneme. CEN1 is an essential gene, the deletion of which results in the loss of basal bodies, which is likely due to defects in both basal body duplication and basal body maintenance. Analysis of the three other centrins indicates that two of them function at microtubule-rich structures unique to ciliates, whereas the fourth is not expressed under conditions examined in this study, although when artificially expressed it localizes to basal bodies. This study provides evidence that in addition to its previously known function in the duplication of basal bodies, centrin is also important for the integrity of these organelles.

Electron tomographic analysis of somatic cell plate formation in meristematic cells of Arabidopsis preserved by high-pressure freezing.

We have investigated the process of somatic-type cytokinesis in Arabidopsis (Arabidopsis thaliana) meristem cells with a three-dimensional resolution of approximately 7 nm by electron tomography of high-pressure frozen/freeze-substituted samples. Our data demonstrate that this process can be divided into four phases: phragmoplast initials, solid phragmoplast, transitional phragmoplast, and ring-shaped phragmoplast. Phragmoplast initials arise from clusters of polar microtubules (MTs) during late anaphase. At their equatorial planes, cell plate assembly sites are formed, consisting of a filamentous ribosome-excluding cell plate assembly matrix (CPAM) and Golgi-derived vesicles. The CPAM, which is found only around growing cell plate regions, is suggested to be responsible for regulating cell plate growth. Virtually all phragmoplast MTs terminate inside the CPAM. This association directs vesicles to the CPAM and thereby to the growing cell plate. Cell plate formation within the CPAM appears to be initiated by the tethering of vesicles by exocyst-like complexes. After vesicle fusion, hourglass-shaped vesicle intermediates are stretched to dumbbells by a mechanism that appears to involve the expansion of dynamin-like springs. This stretching process reduces vesicle volume by approximately 50%. At the same time, the lateral expansion of the phragmoplast initials and their CPAMs gives rise to the solid phragmoplast. Later arriving vesicles begin to fuse to the bulbous ends of the dumbbells, giving rise to the tubulo-vesicular membrane network (TVN). During the transitional phragmoplast stage, the CPAM and MTs disassemble and then reform in a peripheral ring phragmoplast configuration. This creates the centrifugally expanding peripheral cell plate growth zone, which leads to cell plate fusion with the cell wall. Simultaneously, the central TVN begins to mature into a tubular network, and ultimately into a planar fenestrated sheet (PFS), through the removal of membrane via clathrin-coated vesicles and by callose synthesis. Small secondary CPAMs with attached MTs arise de novo over remaining large fenestrae to focus local growth to these regions. When all of the fenestrae are closed, the new cell wall is complete. Few endoplasmic reticulum (ER) membranes are seen associated with the phragmoplast initials and with the TVN cell plate that is formed within the solid phragmoplast. ER progressively accumulates thereafter, reaching a maximum during the late PFS stage, when most cell plate growth is completed.