Asymmetric partitioning of transfected DNA during mammalian cell division.

Foreign DNA molecules and chromosomal fragments are generally eliminated from proliferating cells, but we know little about how mammalian cells prevent their propagation. Here, we show that dividing human and canine cells partition transfected plasmid DNA asymmetrically, preferentially into the daughter cell harboring the young centrosome. Independently of how they entered the cell, most plasmids clustered in the cytoplasm. Unlike polystyrene beads of similar size, these clusters remained relatively immobile and physically associated to endoplasmic reticulum-derived membranes, as revealed by live cell and electron microscopy imaging. At entry of mitosis, most clusters localized near the centrosomes. As the two centrosomes split to assemble the bipolar spindle, predominantly the old centrosome migrated away, biasing the partition of the plasmid cluster toward the young centrosome. Down-regulation of the centrosomal proteins Ninein and adenomatous polyposis coli abolished this bias. Thus, we suggest that DNA clustering, cluster immobilization through association to the endoplasmic reticulum membrane, initial proximity between the cluster and centrosomes, and subsequent differential behavior of the two centrosomes together bias the partition of plasmid DNA during mitosis. This process leads to their progressive elimination from the proliferating population and might apply to any kind of foreign DNA molecule in mammalian cells. Furthermore, the functional difference of the centrosomes might also promote the asymmetric partitioning of other cellular components in other mammalian and possibly stem cells.

The tumour suppressor DiRas3 interacts with C-RAF and downregulates MEK activity to restrict cell migration.

BACKGROUND INFORMATION: The mitogenic pathway, composed of RAF kinases, mitogen-activated protein kinase kinases (MEK) and extracellular signal-regulated kinases (ERK), promotes cell proliferation and migration and is upregulated in many tumours. DiRas3 (ARHI, Noey2), a mainly GTP-bound Ras-like protein with an unusual N-terminal extension, is predominantly lost in ovarian and breast cancers. Its re-expression in these tissues impairs cell proliferation, autophagy, apoptosis and cell migration. Further, loss of DiRas3 correlates with an increase in growth factor-induced ERK phosphorylation. Therefore, DIRAS3 proves to be a curious gene with remarkable tumour suppressing capabilities. However, how DiRas3 interferes with ERK phosphorylation, has remained unknown. RESULTS: We demonstrate that DiRas3 associates in vivo with C-RAF and directly binds in vitro to C-RAF, which is upstream of MEK and ERK. Direct binding of DiRas3 to C-RAF is nucleotide independent, and DiRas3's N-terminal extension alone is not sufficient for binding C-RAF. DiRas3 expression inhibits the activating phosphorylations of MEK and ERK. Serum-induced recruitment of DiRas3 to the plasma membrane depends mainly on its N-terminal extension and less on its C-terminus, bound nucleotide or the presence of Ras-GTP. Correspondingly, removal of the N-terminal extension strongly decreases DiRas3's inhibition of MEK and ERK phosphorylations. Tyrosyl-phosphatases do not contribute significantly to reduction of ERK-phosphorylation byDiRas3. Consistently, downregulation of DiRas3 results in a small but significant and persistent increase in MEK and ERK phosphorylation, but does not increase phosphorylation of P38, AKT and c-Jun NH2-terminal kinase. Finally, downregulation of DiRas3 causes increased cell migration, through a mechanism that is MEK dependent. CONCLUSIONS: These results support a model in which serum signals induce the recruitment of DiRas3 to the plasma membrane, where it is tethered via its N- and C-termini. At the plasma membrane, DiRas3 interacts with C-RAF to specifically suppress the activating phosphorylations on MEK and ERK, thus restricting migration of non-cancer cells. This effect is relatively small, but it is also persistent, suggesting that it contributes to the maintenance of the non-migratory phenotype of non-cancerous tissues, in which DiRas3 is expressed.

The tumor suppressor DiRas3 forms a complex with H-Ras and C-RAF proteins and regulates localization, dimerization, and kinase activity of C-RAF.

The maternally imprinted Ras-related tumor suppressor gene DiRas3 is lost or down-regulated in more than 60% of ovarian and breast cancers. The anti-tumorigenic effect of DiRas3 is achieved through several mechanisms, including inhibition of cell proliferation, motility, and invasion, as well as induction of apoptosis and autophagy. Re-expression of DiRas3 in cancer cells interferes with the signaling through Ras/MAPK and PI3K. Despite intensive research, the mode of interference of DiRas3 with the Ras/RAF/MEK/ERK signal transduction is still a matter of speculation. In this study, we show that DiRas3 associates with the H-Ras oncogene and that activation of H-Ras enforces this interaction. Furthermore, while associated with DiRas3, H-Ras is able to bind to its effector protein C-RAF. The resulting multimeric complex consisting of DiRas3, C-RAF, and active H-Ras is more stable than the two protein complexes H-Ras·C-RAF or H-Ras·DiRas3, respectively. The consequence of this complex formation is a DiRas3-mediated recruitment and anchorage of C-RAF to components of the membrane skeleton, suppression of C-RAF/B-RAF heterodimerization, and inhibition of C-RAF kinase activity.

A dedicated cytoplasmic container collects extrachromosomal DNA away from the mammalian nucleus.

Expression from transfected plasmid DNA is generally transient, but it is unclear what process terminates it. We show that DNA entering mammalian cells is rapidly surrounded by a double membrane in the cytoplasm, in some cases after leaving the nucleus. This cytoplasmic container, termed exclusome, frequently also contains extrachromosomal telomeric DNA, and is maintained by the cell over several division cycles. The exclusome envelope contains endoplasmic reticulum proteins and the inner-nuclear membrane proteins Lap2ß and Emerin, but differs from the nuclear envelope by its fenestrations and the absence of the Lamin B Receptor and nuclear pore complexes. Reduction of exclusome frequency upon overexpressing Emerin's LEM-domain suggests a role for Emerin in plasmid DNA compartmentalization. Thus, cells distinguish extrachromosomal DNA and chromosomes and wrap them into similar yet distinct envelopes keeping the former in the exclusome but the latter in the nucleus, where transcription occurs.

Asymmetric division events promote variability in cell cycle duration in animal cells and Escherichia coli.

Asymmetric cell division is a major mechanism generating cell diversity. As cell cycle duration varies among cells in mammalian tissue culture cells, we asked whether their division asymmetry contributes to this variability. We identify among sibling cells an outlier using hierarchical clustering on cell cycle durations of granddaughter cells obtained by lineage tracking of single histone2B-labelled MDCKs. Remarkably, divisions involving outlier cells are not uniformly distributed in lineages, as shown by permutation tests, but appear to emerge from asymmetric divisions taking place at non-stochastic levels: a parent cell influences with 95% confidence and 0.5% error the unequal partitioning of the cell cycle duration in its two progenies. Upon ninein downregulation, this variability propagation is lost, and outlier frequency and variability in cell cycle durations in lineages is reduced. As external influences are not detectable, we propose that a cell-autonomous process, possibly involved in cell specialisation, determines cell cycle duration variability.

Actin dependence of polarized receptor recycling in Madin-Darby canine kidney cell endosomes.

Mammalian epithelial cell plasma membrane domains are separated by junctional complexes supported by actin. The extent to which actin acts elsewhere to maintain cell polarity remains poorly understood. Using latrunculin B (Lat B) to depolymerize actin filaments, several basolateral plasma membrane proteins were found to lose their polarized distribution. This loss of polarity did not reflect lateral diffusion through junctional complexes because a low-density lipoprotein receptor mutant lacking a functional endocytosis signal remained basolateral after Lat B treatment. Furthermore, Lat B treatment did not facilitate membrane diffusion across the tight junction as observed with ethylenediaminetetraacetic acid or dimethyl sulfoxide treatment. Detailed analysis of transferrin recycling confirmed Lat B depolarized recycling of transferrin from endosomes to the basolateral surface. Kinetic analysis suggested sorting was compromised at both basolateral early endosomes and perinuclear recycling endosomes. Despite loss of function, these two endosome populations remained distinct from each other and from early endosomes labeled by apically internalized ligand. Furthermore, apical and basolateral early endosomes were functionally distinct populations that directed traffic to a single common recycling endosomal compartment even after Lat B treatment. Thus, filamentous actin may help to guide receptor traffic from endosomes to the basolateral plasma membrane.

An inverted microcontact printing method on topographically structured polystyrene chips for arrayed micro-3-D culturing of single cells.

With the goal to investigate the relation of shape and function of single cells or clusters of cells in a 3-dimensional (3-D) microenvironment, we present a novel platform technology to create arrays of microwells on polystyrene (PS) chips for hosting cells in a local microenvironment characterized by controlled shape and surface chemistry. The micro-3-D cell culturing combines 2-dimensional chemical patterning with topographical microstructuring presenting to the cells a local 3-D host structure. Microwells of controlled dimensions were produced by a two-step replication process, based on standard microfabrication of Si, replica molding into poly(dimethylsiloxane), and hot embossing of PS. This allowed the production of large numbers of microstructured surfaces with high reproducibility and fidelity of replication. Using inverted micro contact printing, the plateau surface between the microwells was successfully passivated to block adsorption of proteins and prevent cell attachment by transfer of a graft-copolymer, poly(l-lysine)-g-poly(ethylene glycol). The surface inside the microwells was subsequently modified by spontaneous adsorption of proteins or functionalized PLL-g-PEG/PEG-X (X=biotin or specific, cell-interactive peptide) to elicit specific responses inside the wells. Preliminary cell experiments demonstrated the functionality of such a device to host single epithelial cells (MDCK II) inside the functionalized microwells and thus to control their 3-D shape. This novel platform is useful for fundamental cell-biological studies and applications in the area of cell-based sensing.

Tissue-culture light sheet fluorescence microscopy (TC-LSFM) allows long-term imaging of three-dimensional cell cultures under controlled conditions.

Fluorescence long-term imaging of cellular processes in three-dimensional cultures requires the control of media supply, temperature, and pH, as well as minimal photodamage. We describe a system based on a light sheet fluorescence microscope (LSFM), which is optimized for long-term, multi-position imaging of three-dimensional in-gel cell cultures. The system integrates a stable culture condition control system in the optical path of the light-sheet microscope. A further essential element is a biocompatible agarose container suitable for the LSFM, in which any cell type can be cultured in different gel matrices. The TC-LSFM allows studying any in vitro cultured cell type reacting to, dividing in, or migrating through a three-dimensional extracellular matrix (ECM) gel. For this reason we called it "tissue culture-LSFM" (TC-LSFM). The TC-LSFM system allows fast imaging at multiple locations within a millimeter-sized ECM gel. This increases the number of analyzed events and allows testing population effects. As an example, we show the maturation of a cyst of MDCK (canine kidney epithelial) cells over a period of three days. Moreover, we imaged, tracked, and analyzed MDCK cells during the first five days of cell aggregate formation and discovered a remarkable heterogeneity in cell cycle lengths and an interesting cell death pattern. Thus, TC-LSFM allows performing new long-term assays assessing cellular behavior in three-dimensional ECM-gel cultures. For example migration, invasion or differentiation in epithelial cell systems, stem cells, as well as cancer cells can be investigated.

The study of polarisation in single cells using model cell membranes.

The apicobasal polarisation of epithelial cells within an epithelium is critical for its function as a selective barrier. Microenvironmental parameters, including cell-matrix and cell-cell interactions, contribute to the initiation and orientation of this polarity. However, it is often non-trivial to decipher the differential effects of these parameters in a controlled manner using traditional in vitro platforms. A reductionist platform, consisting of E-cadherin coupled onto laterally mobile supported lipid bilayers, was utilised to mimic E-cadherin presentation in the cell membrane. These functionalised bilayers were generated either on flat 2D surfaces or the interior surfaces of round microwells. This platform enabled the study of E-cadherin adhesion and the initiation of polarisation in a controlled environment, where the dimensionality of the microenvironment, type of protein coating and cell shape could be independently studied. A high proportion of single epithelial cells interacted with and clustered cellular E-cadherin in the presence of E-cadherin functionalised bilayers, which was reduced in the presence of integrin-mediated adhesion. The differential response in E-cadherin clustering correlated with the polarisation of E-cadherin and Na,K-ATPase, a reporter for the induction of basolateral polarity. Neither the three-dimensional presentation of E-cadherin nor the cell shape affected E-cadherin clustering or polarisation in single cells. Thus, the mobile presentation of E-cadherin was sufficient to mimic a cell-cell contact and induce basolateral polarisation in single cells.

A mathematical method for the 3D analysis of rotating deformable systems applied on lumen-forming MDCK cell aggregates.

Cell motility contributes to the formation of organs and tissues, into which multiple cells self-organize. However such mammalian cellular motilities are not characterized in a quantitative manner and the systemic consequences are thus unknown. A mathematical tool to decipher cell motility, accounting for changes in cell shape, within a three-dimensional (3D) cell system was missing. We report here such a tool, usable on segmented images reporting the outline of clusters (cells) and allowing the time-resolved 3D analysis of circular motility of these as parts of a system (cell aggregate). Our method can analyze circular motility in sub-cellular, cellular, multi-cellular, and also non-cellular systems for which time-resolved segmented cluster outlines are available. To exemplify, we characterized the circular motility of lumen-initiating MDCK cell aggregates, embedded in extracellular matrix. We show that the organization of the major surrounding matrix fibers was not significantly affected during this cohort rotation. Using our developed tool, we discovered two classes of circular motion, rotation and random walk, organized in three behavior patterns during lumen initiation. As rotational movements were more rapid than random walk and as both could continue during lumen initiation, we conclude that neither the class nor the rate of motion regulates lumen initiation. We thus reveal a high degree of plasticity during a developmentally critical cell polarization step, indicating that lumen initiation is a robust process. However, motility rates decreased with increasing cell number, previously shown to correlate with epithelial polarization, suggesting that migratory polarization is converted into epithelial polarization during aggregate development.

ROCK-mediated contractility, tight junctions and channels contribute to the conversion of a preapical patch into apical surface during isochoric lumen initiation.

Epithelial cells assemble into three-dimensional aggregates to generate lumen-containing organ substructures. Cells therein contact the extracellular matrix with their basal surface, neighbouring cells with their contact surface and the lumen with their apical surface. We investigated the development of single MDCK cells into aggregates with lumen using quantitative live-cell imaging to identify morphogenetic rules for lumen formation. In two-cell aggregates, membrane insertion into the contact surface established a preapical patch (PAP) characterized by the presence of the apical marker gp135, microvilli and the absence of E-cadherin. This PAP originated from a compartment that had hallmarks of an apical recycling endosome, and matured through Brefeldin-A-sensitive membrane trafficking and the establishment of tight junctions around itself. As a result of the activity of water and ion channels, an optically resolvable lumen formed. Initially, this lumen enlarged without changes in aggregate volume or cell number but with decreasing cell volumes. Additionally, the ROCK1/2-myosin-II pathway counteracted PAP and lumen formation. Thus, lumen formation results from PAP establishment, PAP maturation, lumen initiation and lumen enlargement. These phases correlate with distinct cell surface and volume patterns, which suggests that such morphometric parameters are regulated by trafficking, ROCK-mediated contractility and hydrostatic pressure or vice versa.

The Caenorhabditis elegans septin complex is nonpolar.

Septins are conserved GTPases that form heteromultimeric complexes and assemble into filaments that play a critical role in cell division and polarity. Results from budding and fission yeast indicate that septin complexes form around a tetrameric core. However, the molecular structure of the core and its influence on the polarity of septin complexes and filaments is poorly defined. The septin complex of the nematode Caenorhabditis elegans is formed entirely by the core septins UNC-59 and UNC-61. We show that UNC-59 and UNC-61 form a dimer of coiled-coil-mediated heterodimers. By electron microscopy, this heterotetramer appears as a linear arrangement of four densities representing the four septin subunits. Fusion of GFP to the N termini of UNC-59 and UNC-61 and subsequent electron microscopic visualization suggests that the sequence of septin subunits is UNC-59/UNC-61/UNC-61/UNC-59. Visualization of GFP extensions fused to the extremity of the C-terminal coiled coils indicates that these extend laterally from the heterotetrameric core. Together, our study establishes that the septin core complex is symmetric, and suggests that septins form nonpolar filaments.

IQGAP1 stimulates actin assembly through the N-WASP-Arp2/3 pathway.

IQGAP1 is a conserved modular protein overexpressed in cancer and involved in organizing actin and microtubules in motile processes such as adhesion, migration, and cytokinesis. A variety of proteins have been shown to interact with IQGAP1, including the small G proteins Rac1 and Cdc42, actin, calmodulin, beta-catenin, the microtubule plus end-binding proteins CLIP170 (cytoplasmic linker protein) and adenomatous polyposis coli. However, the molecular mechanism by which IQGAP1 controls actin dynamics in cell motility is not understood. Quantitative co-localization analysis and down-regulation of IQGAP1 revealed that IQGAP1 controls the co-localization of N-WASP with the Arp2/3 complex in lamellipodia. Co-immunoprecipitation supports an in vivo link between IQGAP1 and N-WASP. Pull-down experiments and kinetic assays of branched actin polymerization with N-WASP and Arp2/3 complex demonstrated that the C-terminal half of IQGAP1 activates N-WASP by interacting with its BR-CRIB domain in a Cdc42-like manner, whereas the N-terminal half of IQGAP1 antagonizes this activation by association with a C-terminal region of IQGAP1. We propose that signal-induced relief of the autoinhibited fold of IQGAP1 allows activation of N-WASP to stimulate Arp2/3-dependent actin assembly.

Three-dimensional modeling of mechanical forces in the extracellular matrix during epithelial lumen formation.

Mechanical interactions between cells and extracellular matrix (ECM) mediate epithelial cyst formation. This work relies on the combination of numerical modeling with live cell imaging, to piece together a novel nonintrusive method for determining three-dimensional (3D) mechanical forces caused by shape changes of a multicellular aggregate at the early stages of epithelial cyst formation. We analyzed the evolution of Madin-Darby canine kidney cells in 3D cultures using time-lapse microscopy, with type I collagen gel forming the ECM. The evolving 3D interface between the ECM and the cell aggregate was obtained from microscopy images, and the stress on the surface of a proliferating aggregate and in the surrounding ECM was calculated using the finite element method. The viscoelastic properties of the ECM (a needed input for the finite element method solver) were obtained through oscillatory shear flow experiments on a rheometer. For validation purpose, the forces exerted by an aggregate on a force-sensor array were measured and compared against the computational results.

Molecular mechanisms of epithelial polarity: about shapes, forces, and orientation problems.

In a variety of organs, epithelial cells assemble into networks of cysts and tubules. Such structures can be reproduced in vitro. Here the importance of plasma membrane compartmentalization and forces that drive morphogenetic events during cystogenesis are discussed.

Phosphorylation of IQGAP1 modulates its binding to Cdc42, revealing a new type of rho-GTPase regulator.

The Rho-GTPase Cdc42 is important for the establishment and maintenance of epithelial polarity. Signaling from Cdc42 is propagated via its effector molecules that specifically bind to Cdc42 in the GTP-bound form. The cell-cell contact regulator and actin-binding protein IQGAP1 is described as effector of Cdc42 and Rac. Unexpectedly, we show in this study that IQGAP1 bound also directly nucleotide-depleted Cdc42 (Cdc42-ND). This interaction was enhanced in the presence of phosphatase inhibitors and in epithelial cells without cell-cell contacts. Tandem mass spectrometry analysis and immunoprecipitation experiments revealed that IQGAP1 was Ser1443-phosphorylated in vivo, potentially by protein kinase Cepsilon and upon loss of cell-cell contacts. In addition, we identified two independent domains of the IQGAP1 C terminus that bound exclusively Cdc42-ND. These domains interacted with each other, favoring the binding to Cdc42-GTP. Moreover, phosphorylation on Ser1443 strongly inhibited this intramolecular interaction. Thus, we unraveled a molecular mechanism that reveals a novel type of Rho-GTPase regulator. We propose that, depending on its phosphorylation state, IQGAP1 might serve as an effector or sequester nucleotide-free Cdc42 to prevent signaling.