School of Biotechnology

The cell-cycle transition from G 1 to S phase has been difficult to visualize. We have harnessed antiphase oscillating proteins that mark cell-cycle transitions in order to develop genetically encoded fluorescent probes for this purpose. These probes effectively label individual G 1 phase nuclei red and those in S/G 2 / M phases green. We were able to generate cultured cells and transgenic mice constitutively expressing the cell-cycle probes, in which every cell nucleus exhibits either red or green fluorescence. We performed time-lapse imaging to explore the spatiotemporal patterns of cell-cycle dynamics during the epithelial-mesenchymal transition of cultured cells, the migration and differentiation of neural progenitors in brain slices, and the development of tumors across blood vessels in live mice. These mice and cell lines will serve as model systems permitting unprecedented spatial and temporal resolution to help us better understand how the cell cycle is coordinated with various biological events.

Solid tumors require an efficient decision-making mechanism to progress through a gradient of hypoxia. Here, we show that an oxygen-sensory p53 tetramer-octamer switch makes cell decision for survival or death in variable hypoxia. Trapping homo-oligomers from biosynthesis cycle, we found a metastable p53 tetramer in cells. Under the operation of switch, tetramer segregates the p53 character of a tumor suppressor and promoter. The p53 switch generates a pattern of its on-off state in time that is specific to the strength of hypoxia. A bidirectional tetramer-octamer conversion in on state decides the restoration of basal state by forward and programs apoptosis upon the reverse shift via p53-MDM2 loop. However, reversible dimertetramer transitions in off state trigger chaperoning of HIF-1 complex by tetramer in forward and oncogenic gain-of-function by prion-like dimers in reverse direction. Temporal on-off patterns calibrate stabilized p53 pool by defining the abundance of dimer, tetramer and octamer that ultimately decides diverse cellular outcomes in hypoxia. Through multi-chromophore FRET, we further show that chaperoning of HIF-1 may modulate angiogenesis through a possible flip-flop of the p53T-HIF-1 complex upon DNA. Our results demonstrate how p53 can sense oxygen and act upon its homooligomerization states to control cell fate in hypoxic tumors. .

Identifying similar keywords, known as broad matches, is an important task in online advertising that has become a standard feature on all major keyword advertising platforms. Effective broad matching leads to improvements in both relevance and monetization, while increasing advertisers' reach and making campaign management easier. In this paper, we present a learning-based approach to broad matching that is based on exploiting implicit feedback in the form of advertisement clickthrough logs. Our method can utilize arbitrary similarity functions by incorporating them as features. We present an online learning algorithm, Amnesiac Averaged Perceptron, that is highly efficient yet able to quickly adjust to the rapidly-changing distributions of bidded keywords, advertisements and user behavior. Experimental results obtained from (1) historical logs and (2) live trials on a large-scale advertising platform demonstrate the effectiveness of the proposed algorithm and the overall success of our approach in identifying high-quality broad match mappings. Table 5: List of selected features using Feature Selection. See section 5.1 for a description of these features. Online and Amnesia Keyword-id, log2(Contextual CT R kw ), BM1, BM17 Online and No Amnesia Keyword-id, Contextual CT R kw , Contextual CT R kw > Contextual CT R kw , BM12, Binary-Users-query using PMI, BM14, BM4 Batch and Amnesia

Phototaxis and photophobic responses of green algae are mediated by rhodopsins with microbial type chromophores, i.e. all-trans-retinal in the ground state. The green alga Chlamydomonas reinhardtii was recently completely sequenced and the EST (expressed sequence tag) database was made public. We and others detected overlapping partial cDNA sequences that encode two proteins which we termed channelopsins (Chops). The N-terminal half of chop1 (∼300 of 712 amino acids) comprises hypothetical seven-transmembrane segments with sequence similarity to the proton pump bacteriorhodopsin and the chloride pump halorhodopsin. Even though the overall sequence homology is low, several amino acids are conserved that define the retinal-binding site and the H + -transporting network in BR (bacteriorhodopsin). Expression of Chop1, or only the hydrophobic core, in Xenopus laevis oocytes, enriched with retinal, produced a light-gated conductance (maximum at approx. 500 nm), which shows characteristics of a channel [ChR1 (channelrhodopsin-1)] that is selectively permeable for protons. Also ChR2 (737 amino acids) is an ion channel that is switched directly by light and also here the hydrophobic N-terminal half of the protein is sufficient to enable light-sensitive channel activity. The action spectrum is blue-shifted (maximum at approx. 460 nm) with respect to ChR1. In addition to protons, ChR2 is permeable to univalent and bivalent cations. We suggest that ChRs are involved in phototaxis of green algae. We show that heterologous expression of ChR2 is useful to manipulate intracellular pCa or membrane potential of animal cells, simply by illumination.

Photo-activated adenylate cyclase BLUF domain Amoeboflagellate Naegleria gruberi Flavin Tyrosine Tryptophan Color center emission Protein aging Fluorescence quantum distributions Fluorescence excitation spectra Fluorescence quantum yields a b s t r a c t

The unicellular green alga Chlamydomonas reinhardtii is a versatile model for fundamental and biotechnological research. A wide toolset for genetic manipulation has been developed for this alga, but specific modification of nuclear genes is still not routinely possible. Here, we present a nuclear gene targeting strategy for Chlamydomonas that is based on the application of zinc-finger nucleases (ZFNs). Our approach includes 1) design of gene-specific ZFNs using available online tools; 2) evaluation of the designed ZFNs in a Chlamydomonas in situ model system; 3) optimization of ZFN activity by modification of the nuclease domain; and 4) application of the most suitable enzymes for the mutagenesis of an endogenous gene. Initially, we designed a set of ZFNs for targeting the COP3 gene that encodes the light-activated ion channel channelrhodopsin-1. To evaluate the designed ZFNs, we constructed a model strain by inserting a non-functional aminoglycoside 3'phosphotransferase VIII (aphVIII) selection marker interspaced with a short COP3 target sequence into the nuclear genome. Upon co-transformation of this recipient strain with the engineered ZFNs and an aphVIII DNA template, we were able to restore marker activity and select paromomycin resistant (Pm-R) clones with active nucleases. Of these Pm-R clones, 1% contained a modified COP3 locus as well. In cases where cells were co-transformed with a modified COP3 template, the COP3 locus was specifically modified accordingly by homologous recombination between COP3 and the supplied template DNA. We anticipate that this ZFN technology will be useful for studying the functions of individual genes in Chlamydomonas.

Background: Phototropins are UV-A/blue light receptor proteins with two LOV (Light-Oxygen-Voltage) sensor domains at their N terminus and a kinase domain at the C-terminus in photoautotrophic organisms. This is the first research report of a canonical phototropin from marine algae Ostreococcus tauri. Methods: We synthesized core LOV1 (OtLOV1) domain-encoding portion of the phototropin gene of O. tauri, the domain was heterologously expressed, purified and assessed for its spectral properties and dark recovery kinetics by UV-Visible, fluorescence spectroscopy and mutational studies. Quaternary structure characteristics were studied by SEC and glutaraldehyde crosslinking. Results: The absorption spectrum of OtLOV1 lacks the characteristic 361 nm peak shown by other LOV1 domains. It undergoes a photocycle with a dark state recovery time of approximately 30 min (τ = 300.35 s). Native OtLOV1 stayed as dimer in aqueous solution and the dimer formation was light and concentration independent. Mutating isoleucine at 43rd position to valine accelerated the dark recovery time by more than 10-fold. Mutating it to serine reduced sensitivity to blue light, but the dark recovery time remained unaltered. I43S mutation also destabilized the FMN binding to a great extent. Conclusion: The OtLOV1 domain of the newly identified OtPhot is functional and the isoleucine at position 43 of OtLOV1 is the key residue responsible for fine-tuning the domain properties. General significance: This is the first characterized LOV1 domain of a canonical phototropin from a marine alga and spectral properties of the domain are similar to that of the LOV1 domain of higher plants.

The absorption and emission spectroscopic behavior of lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain consisting of a BLUF domain (BLUF = Blue Light sensor Using Flavin) and a cyclase homology domain was studied in the dark, during blue-light exposure and after blue-light exposure at a temperature of 4°C. The BLUF domain photo-cycle dynamics observed for snap-frozen NgPAC2 was lost by lyophilization (no signaling state formation with flavin absorption red-shift). Instead, blue-light photo-excitation of lyophilized NgPAC2 caused sterically restricted Tyr-Tyr cross-linking (o,o 0-ditysosine formation) and partial flavin cofactor reduction.

Background: Microbial rhodopsins in Chlamydomonas are employed for photoorientation and developmental processes. Results: HKR1 is a UVA-absorbing rhodopsin that is bimodally switched between a UV-and a blue light-absorbing isoform with different light colors. Conclusion: The chromophore of the dark-adapted UV state contains a deprotonated Schiff base stabilized by a 13-cis,15-anti conformation. Significance: This is the initial characterization of the first member of a novel rhodopsin family. Rhodopsins are light-activated chromoproteins that mediate signaling processes via transducer proteins or promote active or passive ion transport as ion pumps or directly light-activated channels. Here, we provide spectroscopic characterization of a rhodopsin from the Chlamydomonas eyespot. It belongs to a recently discovered but so far uncharacterized family of histidine kinase rhodopsins (HKRs). These are modular proteins consisting of rhodopsin, a histidine kinase, a response regulator, and in some cases an effector domain such as an adenylyl or guanylyl cyclase, all encoded in a single protein as a two-component system. The recombinant rhodopsin fragment, Rh, of HKR1 is a UVA receptor (max ‫؍‬ 380 nm) that is photoconverted by UV light into a stable blue light-absorbing meta state Rh-Bl (max ‫؍‬ 490 nm). Rh-Bl is converted back to Rh-UV by blue light. Raman spectroscopy revealed that the Rh-UV chromophore is in an unusual 13-cis,15-anti configuration, which explains why the chromophore is deprotonated. The excited state lifetime of Rh-UV is exceptionally stable, probably caused by a relatively unpolar retinal binding pocket, converting into the photoproduct within about 100 ps, whereas the blue form reacts 100 times faster. We propose that the photochromic HKR1 plays a role in the adaptation of behavioral responses in the presence of UVA light.

Channelrhodopsins (ChRs) are light-gated ion channels that control photomovement of microalgae. In optogenetics, ChRs are widely applied for light-triggering action potentials in cells, tissues, and living animals, yet the spectral properties and photocycle of ChR remain obscure. In this study, we cloned a ChR from the colonial alga Volvox carteri, VChR. After electrophysiological characterization in Xenopus oocytes, VChR was expressed in COS-1 cells and purified. Time-resolved UV-visible spectroscopy revealed a pH-dependent equilibrium of two dark species, D 470 /D 480. Laser flashes converted both with ≈ 200 s into major photointermediates P 510 /P 530 , which reverted back to the dark states with ≈ 15-100 ms. Both intermediates were assigned to conducting states. Three early intermediates P 500 /P 515 and P 390 were detected on a ns to s time scale. The spectroscopic and electrical data were unified in a photocycle model. The functional expression of VChR we report here paves the way toward a broader structure/function analysis of the recently identified class of light-gated ion channels. The behavior of microalgae like Chlamydomonas is controlled by primordial eyes. The earliest events that are recorded from these eyes after light excitation are extremely fast photoreceptor currents (1-3) that are carried by Ca 2ϩ and H ϩ under most conditions (3, 4). The currents are generated by channelrhodopsins (ChRs), 4 microbial type rhodopsins with intrinsic ion conductance (5-7). ChRs gain their light sensitivity and gating properties from the covalently bound chromophore alltrans-retinal, which isomerizes after light absorption (8, 9). Studies in Xenopus oocytes and HEK293 cells revealed ChRs to be proton channels, which also conduct cations like Na ϩ , K ϩ , and Ca 2ϩ (5, 6, 10). In these studies, light flashes evoked the ion * This work was supported by Deutsche Forschungsgemeinschaft Grants Sfb

The flagellate Euglena gracilis contains a photoactivated adenylyl cyclase (PAC), consisting of the flavoproteins PACa and PACb. Here we report functional expression of PACs in Xenopus laevis oocytes, HEK293 cells and in Drosophila melanogaster, where neuronal expression yields light-induced changes in behavior. The activity of PACs is strongly and reversibly enhanced by blue light, providing a powerful tool for light-induced manipulation of cAMP in animal cells.

Photosynthetic unicellular algae have a unique visual system. In Chlamydomonas reinhardtii, the pigmented eye comprises the optical system and at least five different rhodopsin photoreceptors. Two of them, the channelrhodopsins, are rhodopsin-ion channel hybrids switched between closed and open states by photoisomerization of the attached retinal chromophore. They promise to become a useful tool for noninvasive control of membrane potential and intracellular ion concentrations.

Microbial-type rhodopsins are found in archaea, prokaryotes, and eukaryotes. Some of them represent membrane ion transport proteins such as bacteriorhodopsin, a light-driven proton pump, or channelrhodopsin-1 (ChR1), a recently identified light-gated proton channel from the green alga Chlamydomonas reinhardtii . ChR1 and ChR2, a related microbial-type rhodopsin from C. reinhardtii , were shown to be involved in generation of photocurrents of this green alga. We demonstrate by functional expression, both in oocytes of Xenopus laevis and mammalian cells, that ChR2 is a directly light-switched cation-selective ion channel. This channel opens rapidly after absorption of a photon to generate a large permeability for monovalent and divalent cations. ChR2 desensitizes in continuous light to a smaller steady-state conductance. Recovery from desensitization is accelerated by extracellular H + and negative membrane potential, whereas closing of the ChR2 ion channel is decelerated by intracell...

The eyespot of Chlamydomonas reinhardtii is a light-sensitive organelle important for phototactic orientation of the alga. Here, we found that eyespot size is strain specific and downregulated in light. In a strain in which the blue light photoreceptor phototropin was deleted by homologous recombination, the light regulation of the eyespot size was affected. We restored this dysfunction in different phototropin complementation experiments. Complementation with the phototropin kinase fragment reduced the eyespot size, independent of light. Interestingly, overexpression of the N-terminal light, oxygen or voltage sensing domains (LOV1+LOV2) alone also affected eyespot size and phototaxis, suggesting that aside from activation of the kinase domain, they fulfill an independent signaling function in the cell. Moreover, phototropin is involved in adjusting the level of channelrhodopsin-1, the dominant primary receptor for phototaxis within the eyespot. Both the level of channelrhodopsin-1 at the onset of illumination and its steady state level during the light period are downregulated by phototropin, whereas the level of channelrhodopsin-2 is not significantly altered. Furthermore, a light intensity-dependent formation of a C-terminal truncated phototropin form was observed. We propose that phototropin is a light regulator of phototaxis that desensitizes the eyespot when blue light intensities increase.

Inherited mutations of SCN5A, the gene that encodes Na V 1.5, the alpha subunit of the principle voltage-gated Na + channel in the heart, cause congenital Long QT Syndrome variant 3 (LQT-3) by perturbation of channel inactivation. LQT-3 mutations induce small, but aberrant, inward current that prolongs the ventricular action potential and subjects mutation carriers to arrhythmia risk dictated in part by the biophysical consequences of the mutations. Most previously investigated LQT-3 mutations are associated with increased arrhythmia risk during rest or sleep. Here we report a novel LQT-3 mutation discovered in a pediatric proband diagnosed with LQTS but who experienced cardiac events during periods of mild exercise as well as rest. The mutation, which changes a single amino acid (S1904L) in the Na V 1.5 carboxy terminal domain, disrupts the channel inactivation gate complex and promotes late Na + channel currents, not by promoting a bursting mode of gating, but by increasing the propensity of the channel to reopen during prolonged depolarization. Incorporating a modified version of the Markov model of the Na V 1.5 channel into a mathematical model of the human ventricular action potential predicts that the biophysical consequences of the S1904L mutation result in action potential prolongation that is seen for all heart rates but, in contrast to other previously-investigated LQT-3 mutant channels, is most pronounced at fast rates resulting in a drastic reduction in the cells ability to adapt APD to heart rate.

GTPases of the Ras superfamily regulate a wide variety of cellular processes including vesicular transport and various secretory pathways of the cell. ADPribosylation factor (ARF) belongs to one of the five major families of the Ras superfamily and serves as an important component of vesicle formation and transport machinery of the cells. The binding of GTP to these Arfs and its subsequent hydrolysis, induces conformational changes in these proteins leading to their enzymatic activities. The dimeric form of Arf is associated with membrane pinch-off during vesicle formation. In this report, we have identified an arf gene from the unicellular green alga Chlamydomonas reinhardtii, CrArf, and showed that the oligomeric state of the protein in C. renhardtii is modulated by the cellular membrane environment of the organism. Protein cross-linking experiments showed that the purified recombinant CrArf has the ability to form a dimer. Both the 20-kDa monomeric and 40-kDa dimeric forms of CrArf were recognized from Chlamydomonas total cell lysate (CrTLC) and purified recombinant CrArf by the CrArf specific antibody. The membranous environment of the cell appeared to facilitate dimerization of the CrArf, as dimeric form was found exclusively associated with the membrane bound organelles. The subcellular localization studies in Chlamydomonas suggested that CrArf mainly localized in the cytosol and was mislocalized in vesicle transport machinery inhibitor treated cells. This research sheds light on the importance of the cellular membrane environment for regulating the oligomeric state of CrArf protein in this organism and associated functional role.