Summary Cilia use microtubule-based intraflagellar transport (IFT) to organize intercellular signaling. The ciliopathies are a spectrum of human disease resulting from defects in cilia structure or function. Mechanisms regulating assembly of ciliary multiprotein complexes and their transport to the base of cilia remain largely unknown. Combine proteomics, in vivo imaging, and genetic analysis of proteins linked to planar cell polarity (Inturned, Fuzzy, WDPCP), we identified and characterized a new genetic module, which we term CPLANE (ciliogenesis and planar polarity effector) and an extensive associated protein network. CPLANE proteins physically and functionally interact with the poorly understood ciliopathy protein Jbts17 at basal bodies, where they act to recruit a specific subset of IFT-A proteins. In the absence of CPLANE, defective IFT-A particles enter the axoneme, and IFT-B trafficking is severely perturbed. Accordingly, mutation of CPLANE genes elicits specific ciliopathy phenotypes in mouse models and is associated with novel ciliopathies in human patients.

Dosage compensation ensures equal expression of X-linked genes in males and females. In Drosophila, equalization is achieved by hypertranscription of the male X chromosome. This process requires an RNA/protein containing dosage compensation complex (DCC). Here we use RNA interference of individual DCC components to define the order of complex assembly in Schneider cells. We show that interaction of MOF with MSL-3 leads to specific acetylation of MSL-3 at a single lysine residue adjacent to one of its chromodomains. We observe that localization of MSL-3 to the X chromosome is RNA dependent and acetylation sensitive. We find that the acetylation status of MSL-3 determines its interaction with roX2 RNA. Furthermore, we find that RPD3 interacts with MSL-3 and that MSL-3 can be deacetylated by the RPD3 complex. We propose that regulated acetylation of MSL-3 may provide a mechanistic explanation for spreading of the dosage compensation complex along the male X chromosome.

The peroxyoxalate reaction is utilized in a wide variety of analytical applications; however, its mechanism is still not very well understood, especially with respect to the excitation step, where the 'chemical energy' is transformed into 'excitation energy'. This base-catalysed reaction of activated oxalic phenyl esters with hydrogen peroxide in the presence of highly fluorescent aromatic hydrocarbons with low oxidation potentials is the only known chemiluminescence system for which exists experimental evidence for the occurrence of the intermolecular chemically initiated electron exchange luminescence (CIEEL) mechanism of proven high efficiency for excited state formation. We report here the singlet quantum yields and relative rate constants of the excitation step (k(CAT)/k(D)), obtained in the peroxyoxalate reaction, utilizing steroid-substituted oxazolinylidenes as activators. In agreement with the CIEEL mechanism, a linear correlation of ln(k(CAT)/k(D)) with the oxidation potential of the activators is obtained, and the singlet quantum yields can be rationalized in terms of the free energy balance of the back electron transfer, leading to the formation of the activator's excited state. Thus, these results contribute to the experimental validation of the widely employed, thus still controversial, CIEEL mechanism.

The feasibility of applying multiphoton excitation fluorescence microscopy-related techniques in planar membrane systems, such as lipid monolayers at the air-water interface (named Langmuir films), is presented and discussed in this paper. The non-linear fluorescence microscopy approach, allows obtaining spatially and temporally resolved information by exploiting the fluorescent properties of particular fluorescence probes. For instance, the use of environmental sensitive probes, such as LAURDAN, allows performing measurements using the LAURDAN generalized polarization function that in turn is sensitive to the local lipid packing in the membrane. The fact that LAURDAN exhibit homogeneous distribution in monolayers, particularly in systems displaying domain coexistence, overcomes a general problem observed when "classical" fluorescence probes are used to label Langmuir films, i.e. the inability to obtain simultaneous information from the two coexisting membrane regions. Also, the well described photoselection effect caused by excitation light on LAURDAN allows: (i) to qualitative infer tilting information of the monolayer when liquid condensed phases are present and (ii) to provide high contrast to visualize 3D membranous structures at the film's collapse pressure. In the last case, computation of the LAURDAN GP function provides information about lipid packing in these 3D structures. Additionally, LAURDAN GP values upon compression in monolayers were compared with those obtained in compositionally similar planar bilayer systems. At similar GP values we found, for both DOPC and DPPC, a correspondence between the molecular areas reported in monolayers and bilayers. This correspondence occurs when the lateral pressure of the monolayer is 26+/-2 mN/m and 28+/-3 mN/m for DOPC and DPPC, respectively.

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Clonal T cell expansion through proliferation is a central process of the adaptive immune response. Apoptosis of activated T cells is required to avoid chronic inflammation. T cell proliferation and apoptosis are often analyzed with stimuli that do not induce formation of a functional immunological synapse. Here we analyze the Ca 2+ dependence of proliferation and apoptosis in primary human CD4 + T cells following stimulation with anti-CD3/anti-CD28-coated beads, which induce a tight interaction similar to the immunological synapse. We found this focal stimulation to be much more efficient for stimulating IL-2 production and proliferation than non-focal TCR stimuli. Surprising little Ca 2+ entry through Ca 2+ channels was required for T cell proliferation. Transient free intracellular calcium concentration ([Ca 2+ ] i ) elevations of up to 220 nM from a baseline level of around 40 nM were sufficient for maximal proliferation in primary human CD4 + T cells. We also show that proliferation was very Ca 2+ sensitive in the range 90-120 nM, whereas apoptosis was basically constant for [Ca 2+ ] i levels of 90-120 nM. We conclude that very small changes in [Ca 2+ ] i can dramatically change the ratio between proliferation and apoptosis, thus keeping the balance between overshooting and inefficient immune responses.

A recent study on an Asian population reported a six-nucleotide insertion-deletion polymorphism (-652 6N del) in the CASP8 promoter region to be strongly associated with a decreased risk of multiple types of cancer, including breast cancer (BC). Here, we investigate the effect of this deletion in four independent large European BC case-control studies, including data from a total of 7,753 cases and 7,921 controls. The combined per allele odds ratio (OR) was 0.97 (95% confidence interval (CI), 95% CI = 0.93-1.02). The present result indicates that the CASP8 -652 6N del variant has no significant effect on BC risk in Europeans.

Commercial requirements for miniaturized microelectronic devices provide strong motivation for exploring the synthesis of nanoscale systems using bottom-up techniques. The manipulation of charges at the single-electron level in regularly arranged nanoparticles can be utilized to create devices, such as switches, transistors, and digital electronic circuits.[1] An interesting feature of nanoscale ring systems is the AharonovBohm effect: interference phenomena of electron wave functions observed for electrons in ring systems. [2] Up to now, the bottom-up wet-chemical synthesis of inorganic materials has provided a tool for the fabrication of a range of nanostructures, from particles to one-dimensional (1D) structures, [3,4] but these chemical techniques offer little control over the deposition of metals or metal particles as nanometer-sized ring structures, even though interesting properties are expected for such structures. The growth of inorganic materials on biomolecular templates as well as biomolecule-directed assembly of inorganic building blocks are other promising strategies for fabricating complex functional nanostructures, such as wires and ring structures. [5][6][7][8][9] For example, wild-type and genetically engineered viruses have been used to template quasi-1D magnetic [10] and semiconducting nanowires [11] as well as ring structures of Au nanoparticles. [12] In this communication, we address the template-directed deposition of metals on ring-shaped tubulin assemblies for synthesizing metal ring nanostructures. The controlled deposition of metals on polymorphic tubulin structures, as already shown for microtubules (MTs), [13] have great potential for use in future electronic components, for example, AharonovBohm rings to be used in nanoscale circuits. Tubulin is a protein that forms a,b-heterodimers that are about 8 nm in length with diameters ranging from 4 to 5 nm. Tubulin molecules exhibit chemically active surfaces with defined patterns of amino acid side chains, which provide a wide variety of active sites for derivatization, especially for nucleation, organization, and binding metal particles. Tubulin is able to self-assemble into a wide variety of polymorphs: tubules, sheets, ribbons, spirals, and rings, [14] all consisting of differently arranged protofilaments with a strict alternation of a-and b-tubulin monomers. In the presence of Ca 2+ ions, tubulin assembles in vitro into rings and spirals instead of MTs. [15,16] Unlike tubulin rings, MTs have been used as templates to generate metal nanoparticles and nanowires, [13,[17][18][19] for the mineralization of iron oxide, [20] as well as for the kinesinbased transport of synthetic cargo [21][22][23] and CdSe quantum dots.[24]We have investigated ring and spiral formation by transmission electron microscopy (TEM, Fig. 1a). TEM images of tubulin rings fixed by 0.1 % glutaric dialdehyde (GA) and negatively stained by uranyl acetate reveal an outer and inner diameter of 56.4 ± 4.0 nm and 27.1 ± 3.8 nm, respectively, corresponding to a wall thickne...