Bbof1 is required to maintain cilia orientation.

Multiciliate cells (MCCs) are highly specialized epithelial cells that employ hundreds of motile cilia to produce a vigorous directed flow in a variety of organ systems. The production of this flow requires the establishment of planar cell polarity (PCP) whereby MCCs align hundreds of beating cilia along a common planar axis. The planar axis of cilia in MCCs is known to be established via the PCP pathway and hydrodynamic cues, but the downstream steps required for cilia orientation remain poorly defined. Here, we describe a new component of cilia orientation, based on the phenotypic analysis of an uncharacterized coiled-coil protein, called bbof1. We show that the expression of bbof1 is induced during the early phases of MCC differentiation by the master regulator foxj1. MCC differentiation and ciliogenesis occurs normally in embryos where bbof1 activity is reduced, but cilia orientation is severely disrupted. We show that cilia in bbof1 mutants can still respond to patterning and hydrodynamic cues, but lack the ability to maintain their precise orientation. Misexpression of bbof1 promotes cilia alignment, even in the absence of flow or in embryos where microtubules and actin filaments are disrupted. Bbof1 appears to mediate cilia alignment by localizing to a polar structure adjacent to the basal body. Together, these results suggest that bbof1 is a basal body component required in MCCs to align and maintain cilia orientation in response to flow.

Cadherin point mutations alter cell sorting and modulate GTPase signaling.

This study investigated the impact of cadherin binding differences on both cell sorting and GTPase activation. The use of N-terminal domain point mutants of Xenopus C-cadherin enabled us to quantify binding differences and determine their effects on cadherin-dependent functions without any potential complications arising as a result of differences in cytodomain interactions. Dynamic cell-cell binding measurements carried out with the micropipette manipulation technique quantified the impact of these mutations on the two-dimensional binding affinities and dissociation rates of cadherins in the native context of the cell membrane. Pairwise binding affinities were compared with in vitro cell-sorting specificity and ligation-dependent GTPase signaling. Two-dimensional affinity differences greater than five-fold correlated with cadherin-dependent in vitro cell segregation, but smaller differences failed to induce cell sorting. Comparison of the binding affinities with GTPase signaling amplitudes further demonstrated that differential binding also proportionally modulates intracellular signaling. These results show that differential cadherin affinities have broader functional consequences than merely controlling cell-cell cohesion.

Emi2 enables centriole amplification during multiciliated cell differentiation.

Massive centriole amplification during multiciliated cell (MCC) differentiation is a notable example of organelle biogenesis. This process is thought to be enabled by a derived cell cycle state, but the key cell cycle components required for centriole amplification in MCC progenitors remain poorly defined. Here, we show that emi2 (fbxo43) expression is up-regulated and acts in MCC progenitors after cell cycle exit to transiently inhibit anaphase-promoting complex/cyclosome (APC/C)cdh1 activity. We find that this inhibition is required for the phosphorylation and activation of a key cell cycle kinase, plk1, which acts, in turn, to promote different steps required for centriole amplification and basal body formation, including centriole disengagement, apical migration, and maturation into basal bodies. This emi2-APC/C-plk1 axis is also required to down-regulate gene expression essential for centriole amplification after differentiation is complete. These results identify an emi2-APC/C-plk1 axis that promotes and then terminates centriole assembly and basal body formation during MCC differentiation.

Mechanical strain breaks planar symmetry in embryonic epithelia via polarized microtubules.

Mechanical strain can act as a global cue to orient the core planar cell polarity pathway (Fz-PCP) in developing epithelia, but how strain directs a Fz-PCP vector is not known. Here we use live cell imaging of apical microtubules (MTs) and components of the Fz-PCP pathway to analyze epithelial cells in Xenopus embryos as they respond to anisotropic mechanical strain and form a Fz-PCP axis. We find that a Fz-PCP axis can be detected approximately 40 min after the application of strain. By contrast, the density and length of apical MTs increases rapidly (5-10 min) in response to strain, independently of Fz-PCP. These early-forming apical MTs are planar polarized: they align to the strain axis and display a marked bias in plus-end orientation that invariably points towards the cell edge opposite the direction of strain application. We show that these MTs can promote the vectorial transport of Dvl3-GFP containing vesicles along the apical surface in a directed manner, perhaps explaining why PCP signaling fails when MTs are disrupted. Finally, we provide evidence that the Fz-PCP axis feeds back after an hour to stabilize oriented apical MTs. These results provide insights into how mechanical strain acts as a developmental cue within the appropriate time frame and with the appropriate vector to promote planar axis formation.

Memory in receptor-ligand-mediated cell adhesion.

Single-molecule biomechanical measurements, such as the force to unfold a protein domain or the lifetime of a receptor-ligand bond, are inherently stochastic, thereby requiring a large number of data for statistical analysis. Sequentially repeated tests are generally used to obtain a data ensemble, implicitly assuming that the test sequence consists of independent and identically distributed (i.i.d.) random variables, i.e., a Bernoulli sequence. We tested this assumption by using data from the micropipette adhesion frequency assay that generates sequences of two random outcomes: adhesion and no adhesion. Analysis of distributions of consecutive adhesion events revealed violation of the i.i.d. assumption, depending on the receptor-ligand systems studied. These include Markov sequences with positive (T cell receptor interacting with antigen peptide bound to a major histocompatibility complex) or negative (homotypic interaction between C-cadherins) feedbacks, where adhesion probability in the next test was increased or decreased, respectively, by adhesion in the immediate past test. These molecular interactions mediate cell adhesion and cell signaling. The ability to "remember" the previous adhesion event may represent a mechanism by which the cell regulates adhesion and signaling.

Mechanical Strain Determines Cilia Length, Motility, and Planar Position in the Left-Right Organizer.

The Xenopus left-right organizer (LRO) breaks symmetry along the left-right axis of the early embryo by producing and sensing directed ciliary flow as a patterning cue. To carry out this process, the LRO contains different ciliated cell types that vary in cilia length, whether they are motile or sensory, and how they position their cilia along the anterior-posterior (A-P) planar axis. Here, we show that these different cilia features are specified in the prospective LRO during gastrulation, based on anisotropic mechanical strain that is oriented along the A-P axis, and graded in levels along the medial-lateral axis. Strain instructs ciliated cell differentiation by acting on a mesodermal prepattern present at blastula stages, involving foxj1. We propose that differential strain is a graded, developmental cue, linking the establishment of an A-P planar axis to cilia length, motility, and planar location during formation of the Xenopus LRO.

Mechanical strain determines the axis of planar polarity in ciliated epithelia.

Epithelia containing multiciliated cells align beating cilia along a common planar axis specified by the conserved planar cell polarity (PCP) pathway. Specification of the planar axis is also thought to require a long-range cue to align the axis globally, but the nature of this cue in ciliated and other epithelia remains poorly understood. We examined this issue using the Xenopus larval skin, where ciliary flow aligns to the anterior-posterior (A-P) axis. We first show that a planar axis initially arises in the developing skin during gastrulation, based on the appearance of polarized apical microtubules and cell junctions with increased levels of stable PCP components. This axis also arises in severely ventralized embryos, despite their deficient embryonic patterning. Because ventralized embryos still gastrulate, producing a mechanical force that strains the developing skin along the A-P axis, we asked whether this strain alone drives global planar patterning. Isolated skin explanted before gastrulation lacks strain and fails to acquire a global planar axis but responds to exogenous strain by undergoing cell elongation, forming polarized apical microtubules, and aligning stable components of the PCP pathway orthogonal to the axis of strain. The planar axis in embryos can be redirected by applying exogenous strain during a critical period around gastrulation. Finally, we provide evidence that apical microtubules and the PCP pathway interact to align the planar axis. These results indicate that oriented tissue strain generated by the gastrulating mesoderm plays a major role in determining the global axis of planar polarity of the developing skin.

Nitric oxide inhibits DNA-adduct excision in nucleotide excision repair.

Sustained induction of nitric oxide (NO) in chronic inflammation may be mutagenic, through DNA damage induction and/or DNA repair inhibition. Although there is good evidence that NO can cause DNA damage, how NO is involved in DNA repair remains elusive. By using DNA synthesis inhibitors to accumulate DNA strand breaks in comet assay, we show that NO and peroxynitrite inhibit DNA-adduct excision in human fibroblasts damaged by UVC, 4-nitroquinoline 1-oxide, benzo[a]pyrene dihydrodiol epoxide, cisplatin, or mitomycin C, but not with methyl methane sulfonate. Treating cells with arsenite increased NO production and also inhibited the DNA-adduct excision induced by UVC, 4-nitroquinoline 1-oxide, benzo[a]pyrene dihydrodiol epoxide, cisplatin, and mitomycin C, but not by methyl methane sulfonate, H(2)O(2), sodium nitrosoprusside, or 3-morpholinosydnonimine. Arsenite inhibition of DNA-adduct excision was decreased by NO synthase inhibitors and NO scavengers. The nuclear extract prepared from fibroblasts pretreated with sodium nitrosoprusside, dipropylenetriamine NONOate, 3-morpholinosydnonimine, or arsenite also showed decreased activity in excising the DNA adducts induced by UVC and cisplatin but not by methyl methane sulfonate or H(2)O(2) plus Fe. These results are consistent with the notion that NO, peroxynitrite, and arsenite inhibit the DNA-adduct excision in nucleotide excision repair but not that in base excision repair.

Biophysical properties of cadherin bonds do not predict cell sorting.

Differential binding between cadherin subtypes is widely believed to mediate cell sorting during embryogenesis. However, a fundamental unanswered question is whether cell sorting is dictated by the biophysical properties of cadherin bonds, or by broader, cadherin-dependent differences in intercellular adhesion or membrane tension. This report describes atomic force microscope measurements of the strengths and dissociation rates of homophilic and heterophilic cadherin (CAD) bonds. Measurements conducted with chicken N-CAD, canine E-CAD, and Xenopus C-CAD demonstrated that all three cadherins cross-react and form multiple, intermolecular bonds. The mechanical and kinetic properties of the heterophilic bonds are similar to the homophilic interactions. The thus quantified bond parameters, together with previously reported adhesion energies were further compared with in vitro cell aggregation and sorting assays, which are thought to mimic in vivo cell sorting. Trends in quantified biophysical properties of the different cadherin bonds do not correlate with sorting outcomes. These results suggest that cell sorting in vivo and in vitro is not governed solely by biophysical differences between cadherin subtypes.

Two stage cadherin kinetics require multiple extracellular domains but not the cytoplasmic region.

Micropipette manipulation measurements quantified the pre-steady state binding kinetics between cell pairs mediated by Xenopus cleavage stage cadherin. The time-dependence of the intercellular binding probability exhibits a fast forming, low probability binding state, which transitions to a slower forming, high probability state. The biphasic kinetics are independent of the cytoplasmic region, but the transition to the high probability state requires the third extracellular domain EC3. Deleting either EC3 or EC3-5, or substituting Trp(2) for Ala reduces the binding curves to a simple, monophasic rise in binding probability to a limiting plateau, as predicted for a single site binding mechanism. The two stage cadherin binding process reported here directly parallels previous biophysical studies, and confirms that the cadherin ectodomain governs the initial intercellular adhesion dynamics.

Comet assay with nuclear extract incubation.

Alkaline comet assay is a simple sensitive method for detecting DNA strand breaks. However, at the time of cell lysis, only a fraction of the entire DNA damage appears as DNA strand breaks, while some DNA strand breaks may have been rejoined and some DNA lesions may still remain unexcised. We showed that nuclear extract (NE) prepared from human cells could excise the DNA adducts induced by UVC, X-ray, and methyl methanesulfonate (MMS). Thus, the comet assay with NE incubation allows a closer estimation of total DNA damage. Among the human urothelial carcinoma cell lines we tested, the NE of NTUB1 cells showed higher activity in excising the DNA adducts induced by UVC, but with a lower activity in excising the DNA adducts induced by MMS than the NE of BFTC905 cells. Moreover, under the same dose of X-ray irradiation, a larger difference in total DNA damage between two cell lines was revealed in comet assay incubated with NE than without NE. Therefore, the comet assay with NE incubation may be useful in the research of cancer risk, drug resistance, and DNA repair proteins.