SV40 Nuclear Localization Signal Research Articles

Designing Fluorescent Nuclear Permeable Peptidomimetics to Target Proliferating Cell Nuclear Antigen.

Human proliferating cell nuclear antigen (PCNA) is a critical mediator of DNA replication and repair, acting as a docking platform for replication proteins. Disrupting these interactions with a peptidomimetic agent presents as a promising avenue to limit proliferation of cancerous cells. Here, a p21-derived peptide was employed as a starting scaffold to design a modular peptidomimetic that interacts with PCNA and is cellular and nuclear permeable. Ultimately, a peptidomimetic was produced which met these criteria, consisting of a fluorescein tag and SV40 nuclear localization signal conjugated to the N-terminus of a p21 macrocycle derivative. Attachment of the fluorescein tag was found to directly affect cellular uptake of the peptidomimetic, with fluorescein being requisite for nuclear permeability. This work provides an important step forward in the development of PCNA targeting peptidomimetics for use as anti-cancer agents or as cancer diagnostics.

Unexploited Performance of NLS in the dCas9-VPR-Mediated Transcriptional Activation.

Nuclear localization signal (NLS) is a short peptide guiding the nuclear transport process, recognized as playing an important role in constructing clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) activators. Here, we investigate the effect of the position and number of the NLS on transcriptional activation based on the dCas9-VPR activator. Our results not only demonstrate that the position of the SV40 NLS could have different degrees of influence on activation efficiency but also, surprisingly, we find that the SV40 NLS plays a detrimental role. Complete deletion of the NLS from the system could increase the transcriptional activation efficiency by 2 to 4 times compared with the original dCas9-VPR. This finding is also supported by some typical first- and third-generation activators. Our work should be beneficial to the design of the NLS-based system.

Bioactivated and PEG-Protected Circa 2nm Gold Nanoparticles for in Cell Labelling and Cryo-Electron Microscopy.

Advances in cryo-electron microscopy (EM) enable imaging of protein assemblies within mammalian cells in a near native state when samples are preserved by cryogenic vitrification. To accompany this progress, specialized EM labelling protocols must be developed. Gold nanoparticles (AuNPs) of 2nm are synthesized and functionalized to bind selected intracellular targets inside living human cells and to be detected in vitreous sections. As a proof of concept, thioaminobenzoate-, thionitrobenzoate-coordinated gold nanoparticles are functionalized on their surface with SV40 Nuclear Localization Signal (NLS)-containing peptides and 2kDa polyethyleneglycols (PEG) by thiolate exchange to target the importin-mediated nuclear machinery and facilitate cytosolic diffusion by shielding the AuNP surface from non-specific binding to cell components, respectively. After delivery by electroporation into the cytoplasm of living human cells, the PEG-coated AuNPs diffuse freely in the cytoplasm but do not enter the nucleus. Incorporation of NLS within the PEG coverage promotes a quick nuclear import of the nanoparticles in relation to the density of NLS onto the AuNPs. Cryo-EM of vitreous cell sections demonstrate the presence of 2nm AuNPs as single entities in the nucleus. Biofunctionalized AuNPs combined with live-cell electroporation procedures are thus potent labeling tools for the identification of macromolecules in cellular cryo-EM.

ABA represses TOR and root meristem activity through nuclear exit of the SnRK1 kinase.

The phytohormone abscisic acid (ABA) promotes plant tolerance to major stresses such as drought, partly by modulating growth through poorly understood mechanisms. Here, we show that ABA-triggered repression of cell proliferation in the Arabidopsis thaliana root meristem relies on the swift subcellular relocalization of SNF1-RELATED KINASE 1 (SnRK1). Under favorable conditions, the SnRK1 catalytic subunit, SnRK1α1, is enriched in the nuclei of root cells, and this is accompanied by normal cell proliferation and meristem size. Depletion of two key drivers of ABA signaling, SnRK2.2 and SnRK2.3, causes constitutive cytoplasmic localization of SnRK1α1 and reduced meristem size, suggesting that, under nonstress conditions, SnRK2s promote growth by retaining SnRK1α1 in the nucleus. In response to ABA, SnRK1α1 translocates to the cytoplasm, and this is accompanied by inhibition of target of rapamycin (TOR), decreased cell proliferation, and reduced meristem size. Blocking nuclear export with leptomycin B abrogates ABA-driven SnRK1α1 relocalization to the cytoplasm and ABA-elicited inhibition of TOR. Furthermore, fusing SnRK1α1 to an SV40 nuclear localization signal leads to defective ABA-dependent TOR repression. Altogether, we demonstrate that SnRK2-dependent changes in SnRK1α1 subcellular localization are crucial for inhibiting TOR and root growth in response to ABA. Rapid relocalization of central regulators such as SnRK1 may represent a general strategy of eukaryotic organisms to respond to environmental changes.

Transgenic mice encoding modern imaging probes: Properties and applications.

SUMMARYModern biology is increasingly reliant on optical technologies, including visualization and longitudinal monitoring of cellular processes. The major limitation here is the availability of animal models to track the molecules and cells in their natural environment in vivo. Owing to the integrity of the studied tissue and the high stability of transgene expression throughout life, transgenic mice encoding fluorescent proteins and biosensors represent unique tools for in vivo studies in norm and pathology. We review the strategies for targeting probe expression in specific tissues, cell subtypes, or cellular compartments. We describe the application of transgenic mice expressing fluorescent proteins for tracking protein expression patterns, apoptotic events, tissue differentiation and regeneration, neurogenesis, tumorigenesis, and cell fate mapping. We overview the possibilities of functional imaging of secondary messengers, neurotransmitters, and ion fluxes. Finally, we provide the rationale and perspectives for the use of transgenic imaging probes in translational research and drug discovery.

Optimization of Cas9 nuclear localization in a tilapia (Oreochromis mossambicus) cell line

CRISPR gene targeting studies in cell lines require reliable expression of Cas9 along with appropriate target‐specific guide RNAs. Effective CRISPR/Cas9 gene editing requires Cas9 localization in the nucleus. Previous attempts to implement CRISPR/Cas9 gene editing in a tilapia (Oreochromis mossambicus) cell line (OmB cells) were unsuccessful. A possible contribution to these results could be inefficient nuclear localization of Cas9. Conventionally, the mammalian SV40 nuclear localization signal (SV40 NLS) is used to localize proteins to the nucleus. Our previous studies have shown that the SV40 promoter is poorly recognized in OmB cells. Therefore, we hypothesized that the SV40 NLS will also be poorly recognized in OmB cells. To test this hypothesis we fused the SV40 NLS to the C‐terminus of GFP (green fluorescent protein), transfected it into the tilapia OmB cell line, and determined the efficiency of nuclear GFP localization using fluorescence imaging (Leica Dmi8). Our imaging results show that the SV40 NLS moderately induced nuclear GFP localization. An incomplete but significant increase in nuclear GFP localization relative to the control (no NLS, just GFP) was observed using Leica image analysis software (LASX 2.0, Leica). Nevertheless, a large amount of GFP still remained in the cytosol when using SV40 NLS. Current work is comparing the efficiency of SV40 NLS mediated nuclear localization of GFP in fish cell lines to that mediated by an endogenous tilapia NLS. We have cloned an endogenous tilapia NLS and are currently testing whether this endogenous NLS improves nuclear localization of ectopic GFP in tilapia OmB cells. Optimization of nuclear localization of Cas9 will aid future gene editing studies in the OmB cell line and in cells from related fish species.Support or Funding InformationThis study was funded by a grant from NSF (IOS‐1656371).

Recombinant Fusion Streptavidin as a Scaffold for DNA Nanotetrads for Nucleic Acid Delivery and Telomerase Activity Imaging in Living Cells.

Efficient platforms for intracellular delivery of nucleic acids are essential for biomedical imaging and gene regulation. We develop a recombinant fusion streptavidin as a novel protein scaffold for DNA nanotetrads for highly efficient nucleic acid delivery and telomerase activity imaging in living cells via cross-linking hybridization chain reaction (cHCR). The recombinant streptavidin protein is designed to fuse with multiple SV40 NLS (nuclear localization signal) and NES (nuclear export signal) domains and prepared through Escherichia coli expression. The recombinant NLS-SA protein allows facile assembly with four biotinylated DNA probes via high-affinity noncovalent interactions, forming a well-defined DNA tetrad nanostructure. The DNA nanotetrads are demonstrated to confer efficient cytosolic delivery of nucleic acid via a caveolar mediated endocytosis pathway, allowing efficient escape from lysosomal degradation. Moreover, the nanotetrads enable efficient cHCR assembly in response to telomerase in vitro and in cellulo, affording ultrasensitive detection and spatially resolved imaging for telomerase with a detection limit as low as 90 HeLa cells/mL. The fluorescence brightness obtained in live cell imaging is found to be dynamically correlated to telomerase activity and the inhibitor concentrations. Therefore, the proposed strategy may provide a highly efficient platform for nucleic acid delivery and imaging of biomarkers in living cells.

A simple and efficient CRISPR/Cas9 platform for induction of single and multiple, heritable mutations in barley (Hordeum vulgare L.)

BackgroundGenome editing of monocot plants can be accomplished by using the components of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated Cas9) technology specifically optimized for these types of plants. Here, we present the development of RNA-guided Cas9 system for simplex and multiplex genome editing in barley.ResultsWe developed a set of customizable RNA-guided Cas9 binary vectors and sgRNA modules for simplex and multiplex editing in barley. To facilitate the design of RNA-guided Cas9 constructs, the pBract derived binary vectors were adapted to Gateway cloning and only one restriction enzyme was required for construction of the sgRNA. We designed a synthetic, codon optimized Cas9 gene containing the N terminal SV40 nuclear localization signal and the UBQ10 Arabidopsis 1st intron. Two different sgRNAs were constructed for simplex editing and one polycistronic tRNA-gRNA construct (PTG) for multiplex editing using an endogenous tRNA processing system. The RNA-guided Cas9 constructs were validated in transgenic barley plants produced by Agrobacterium-mediated transformation. The highest mutation rate was observed in simplex editing of the cytokinin oxidase/dehydrogenase HvCKX1 gene, where mutations at the hvckx1 locus were detected in 88% of the screened T0 plants. We also proved the efficacy of the PTG construct in the multiplex editing of two CKX genes by obtaining 9 plants (21% of all edited plants) with mutations induced in both HvCKX1 and HvCKX3. Analysis of the T1 lines revealed that mutations in the HvCKX1 gene were transmitted to the next generation of plants. Among 220 screened T1 plants we identified 85 heterozygous and 28 homozygous mutants, most of them bearing frameshift mutations in the HvCKX1 gene. We also observed independent segregation of mutations and the Cas9-sgRNA T-DNA insert in several T1 plants. Moreover, the knockout mutations of the Nud gene generated phenotype mutants with naked grains, and the phenotypic changes were identifiable in T0 plants.ConclusionsWe demonstrated the effectiveness of an optimized RNA-guided Cas9 system that can be used for generating homozygous knockout mutants in the progeny of transgenic barely plants. This is also the first report of successful multiplex editing in barley using a tRNA processing system.

T7 Polymerase Expression of Guide RNAs in vivo Allows Exportable CRISPR-Cas9 Editing in Multiple Yeast Hosts.

Efficient guide RNA expression often limits CRISPR-Cas9 implementation in new hosts. To address this limitation in fungal systems, we demonstrate the utility of a T7 polymerase system to effectively express sgRNAs. Initially, we developed a methodology in Saccharomyces cerevisiae using a modified version of the T7 P266L mutant polymerase with an SV40 nuclear localization signal to allow guide RNA expression immediately downstream of a T7 promoter. To improve targeting efficiency, guide RNA design was found to be tolerant to three mismatches or up to three additional bases appended to the 5' end. The addition of three guanines to a T7-based guide RNA improved guide RNA expression 80-fold and achieved transcriptional output similar to the strong Pol III snr52 promoter. Resulting gene editing and dCas9-guided gene regulation with a T7-based guide RNA was on par with the commonly used snr52 system in S.cerevisiae. Finally, 96% and 60% genome editing efficiencies were achieved in Kluyveromyces lactis and Yarrowia lipolytica respectively with minimal optimization of this system. Thus, T7-based expression of sgRNAs offers an orthogonal method for implementing CRISPR systems in fungal systems.

Detection and quantification of dengue virus using a novel biosensor system based on dengue NS3 protease activity.

BackgroundThe traditional methods, plaque assays and immuno-focus assays, used to titrate infectious dengue virus (DENV) particles are time consuming and labor intensive. Here, we developed a DENV protease activity detection system (DENPADS) to visualize DENV infection in cells based on dengue protease activity.Methodology/Principal findingsDengue NS3 protease cleaves NS4B-NS5. BHK-21 cells stably expressing the sensor module comprising DENV-2 NS4 and the 10 amino-terminal amino acids of NS5 (N10NS5) fused with the SV40 nuclear localization signal (NLS) and Cre recombinase (Cre), were generated. Cre is constrained outside the nucleus in the absence of NS3 activity but translocates into the nucleus through NS4B-NS5 cleavage when cells are infected with DENV. Nuclear translocation of Cre can trigger the reporter system, which contains a cis-loxP-flanked mCherry with three continuous stop codons following an SV40 polyA tail cDNA upstream of EGFP or mHRP cDNA. Our results show that DENPADS is an efficient and accurate method to titrate 4 DENV serotypes in 24 hours. Compared with current virus titration methods, the entire process is easy to perform, and the data are easily acquired.Conclusions/SignificanceIn this study, we demonstrate that DENPADS can be used to detect dengue viral infection through a fluorescence switch or HRP activity in the infected cells. This approach is sensitive with less incubation time and labor input. In addition, DENPADS can simultaneously evaluate the efficacy and cytotoxicity of potential anti-DENV candidates. Overall, DENPADS is a useful tool for dengue research.

Arginine-rich cross-linking peptides with different SV40 nuclear localization signal content as vectors for intranuclear DNA delivery

The major barriers for intracellular DNA transportation by cationic polymers are their toxicity, poor endosomal escape and inefficient nuclear uptake. Therefore, we designed novel modular peptide-based carriers modified with SV40 nuclear localization signal (NLS). Core peptide consists of arginine, histidine and cysteine residues for DNA condensation, endosomal escape promotion and interpeptide cross-linking, respectively. We investigated three polyplexes with different NLS content (10 mol%, 50 mol% and 90 mol% of SV40 NLS) as vectors for intranuclear DNA delivery. All carriers tested were able to condense DNA, to protect it from DNAase I and were not toxic to the cells. We observed that cell cycle arrest by hydroxyurea did not affect transfection efficacy of NLS-modified carriers which we confirmed using quantitative confocal microscopy analysis. Overall, peptide carrier modified with 90 mol% of SV40 NLS provided efficient transfection and nuclear uptake in non-dividing cells. Thus, incorporation of NLS into arginine-rich cross-linking peptides is an adequate approach to the development of efficient intranuclear gene delivery vehicles.

Oocyte shuttle, a recombinant protein transporting donor DNA into the Xenopus oocyte in situ.

ABSTRACTThe newly developed oocyte shuttle protein contains a streptavidin moiety that tightly binds biotinylated DNA. Injected intravenously into adult Xenopus females, the protein-DNA complex is rapidly transported through the bloodstream and, within the ovary, the vitellogenin ligand present in the protein binds to the receptors at the surface of the oocytes. The bound complex is internalized and translocates into the oocyte nucleus thanks to an SV40 nuclear localization signal, enhanced by an adjacent casein kinase phosphorylation site. Functioning of the shuttle protein is documented by transporting DNA molecules that, upon intramolecular homologous recombination within the oocyte nucleus, express easily traceable markers such as green fluorescence or tetracycline resistance.

A Doubletime Nuclear Localization Signal Mediates an Interaction with Bride of Doubletime to Promote Circadian Function

Doubletime (DBT) has an essential circadian role in Drosophila melanogaster because it phosphorylates Period (PER). To determine if DBT antagonism can produce distinct effects in the cytosol and nucleus, forms of a dominant negative DBT(K/R) with these 2 alternative localizations were produced. DBT has a putative nuclear localization signal (NLS), and mutation of this signal confers cytosolic localization of DBT in the lateral neurons of Drosophila clock cells in the brain. By contrast, addition of a strong NLS domain (e.g., SV40 NLS) to DBT's C terminus leads to more nuclear localization. Expression of DBT(K/R) with the mutated NLS (DBT(K/R) NLS(-)) using a timGAL4 driver does not alter the circadian period of locomotor activity, and the daily oscillations of PER detected by immunoblot and immunofluorescence persist, like those of wild-type flies. By contrast, expression of DBT(K/R) with the strong NLS (DBT(K/R) stNLS) using the timGAL4 driver lengthens period more strongly than DBT(K/R), with damped oscillations of PER phosphorylation and localization. Both DBT(K/R) and DBT(WT) without the NLS fail to interact with Bride of Doubletime (BDBT) protein, which is related to FK506-binding proteins and shown to interact with DBT to enhance its circadian function. This result suggests that the DBT(K/R) NLS(-) has lost its dominant negative property because it does not form normal clock protein complexes. DBT(WT) proteins with the same changes (NLS(-) and stNLS) also produce equivalent changes in localization that do not produce opposite period phenotypes. Additionally, a DBT(K/R) protein with both the stNLS and NLS(-) mutation does not affect circadian period, although it is nuclear, demonstrating that the lack of a dominant negative for the DBT(K/R) NLS(-) is not due to failure to localize to nuclei. Finally, bdbt RNAi increases the cytosolic localization of DBT(K/R) but not of DBT(WT), suggesting a role for BDBT in DBT kinase-dependent nuclear localization of DBT.

The long N-terminus of the C. elegans DNA repair enzyme APN-1 targets the protein to the nucleus of a heterologous system

We previously isolated from a Caenorhabditis elegans cDNA library, designed for two-hybrid screening, a gene encoding the DNA repair enzyme APN-1 using cross-specie complementation analysis of the Saccharomyces cerevisiae apn1∆ apn2∆ tpp1∆ triple mutant deficient in the ability to repair several types of DNA lesions including apurinic/apyrimidinic (AP) sites. We subsequently purified the APN-1 from this yeast mutant and demonstrated that it possesses four distinct DNA repair activities. However, following the re-annotation of the C. elegans genome we discovered that the functionally active APN-1 encoded by the cDNA from the library might lack 108 amino acid residues from the N-terminus. We therefore synthesized the entire C. elegans apn-1 gene encoding the putative full-length APN-1 and created several N-terminal deletion mutants lacking either 63, 83 or 118 amino acid residues. The full-length APN-1, APN-1 (1–63Δ) and APN-1 (1–83Δ), but not APN-1 (1–118Δ) were stably expressed in the yeast triple mutant and cleaved the AP site substrate. However, only the full-length APN-1 rescued the yeast mutant from the genotoxicity caused by methyl methane sulfonate, a DNA damaging agent that creates AP sites in the genome. The full-length APN-1 was localized to the yeast nucleus, while APN-1 (1–63Δ) and APN-1 (1–83Δ) retained a cytoplasmic distribution. Our data suggest that the N-terminal region has no direct role in the DNA repair functions of APN-1 other than to target the protein to the nucleus and possibly to maintain its stability. Thus, the truncated APN-1, previously isolated from the two-hybrid library, ability to complement the yeast triple mutant depends on the engineered SV40 nuclear localization signal.

Abstract 3319: PKCδ switches from a pro-apoptotic to a pro-survival signal in K-ras “dependent” lung cancer

Abstract Oncogenic mutations in KRAS are found in about 25% of non-small cell lung cancers (NSCLC), however only a subset of these tumors are dependent upon K-ras for survival. We have previously shown that PKCδ is required for transformed growth of NSCLC cells that are dependent on K-ras, and for growth of K-ras driven lung tumors in vivo. PKCδ also plays an essential role in DNA damage-induced apoptosis in many cell types. Analysis of 14 mutant KRAS NSCLC cell lines that are K-ras “independent” for survival (A549, H157, H460, SW1573, H1155, H1792), or K-ras “dependent” (H2009, H727, HCC44, H2122, SW900, H1573, H358, H441) reveals that the pro-tumorigenic and pro-apoptotic functions of PKCδ segregate with the K-ras dependency status, and reflect differential regulation of survival signaling by PKCδ in these two cell populations. K-ras-independent NSCLC cells are sensitive to DNA-damage-induced apoptosis, and shRNA mediated depletion of PKCδ in these cells results in suppression of apoptosis and increased activation of the MEK/ERK pathway. In contrast, K-ras dependent NSCLC cells are resistance to DNA-damage-induced apoptosis and depletion of PKCδ enhances apoptosis and suppresses MEK/ERK activation, anchorage-independent growth, and invasion/migration. To explore why K-ras dependent NSCLC cells are resistant to apoptosis, we analyzed PKCδ Y64 phosphorylation and nuclear translocation, events we have previously shown are critical for the pro-apoptotic function of PKCδ. Surprisingly, K-ras dependent cells have an up to 7-fold increase in the nuclear:cytoplasmic ratio of PKCδ, increased phosphorylation of PKCδ Y64, and increased (1.5-3 fold) PKCδ mRNA and protein, suggesting that these cells may be refractive to PKCδ driven apoptosis. To test this we expressed a mutant of PKCδ targeted to the nucleus by the addition of a SV40 nuclear localization signal. Expression of PKCδNLS induced apoptosis and increased DNA damage-induced apoptosis in K-ras independent but not K-ras dependent NSCLC cells, confirming that the later are unable to activate apoptotic pathways downstream of PKCδ. These studies define two distinct subsets of NSCLC cell lines in the context of mutant KRAS. NSCLC cells that are “independent” of K-ras preferentially utilize PKCδ for apoptosis, while cells “dependent” on oncogenic K-ras preferentially use PKCδ for proliferation and survival. We are currently using two large, publicly available lung cancer cell line gene expression datasets (GEO accession #'s GSE4824 and GSE36133) to identify pathways and signaling networks unique to these two populations of NSCLC cells. Citation Format: Mary E. Reyland, Angela M. Ohm, Michael G. Edwards, Jennifer Symonds. PKCδ switches from a pro-apoptotic to a pro-survival signal in K-ras “dependent” lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3319. doi:10.1158/1538-7445.AM2014-3319

Nanoscale Protein Diffusion by Sted-Based Spatiotemporal Fluorescence Correlation Spectroscopy

Fluorescence Correlation Spectroscopy (FCS) represents an established technique to recover single-molecule diffusion and binding properties in cells. Recently, scanning microscopy imaging was applied to add a spatial dimension to the classic, purely temporal, FCS modality: spatiotemporal FCS (stFCS) provides details about the routes that diffusing particles or molecules follow in the specimen.[1] We report on combining spatiotemporal fluorescence correlation spectroscopy (stFCS) and stimulated emission depletion (STED) to monitor intracellular protein diffusion at a spatial resolution below the optical diffraction limit (super-resolution)[2,3]. We validated our method both in vitro and at intracellular level, studying the diffusion of fluorescent nanocapsids and of GFP bound to SV40 Nuclear Localization Signal (NLS), respectively. NLS-GFP represents a well-known model of actively nuclear-imported protein that has been the subject of intense research.[4] We demonstrated how relevant is our approach discovering persistent complexes between nucleo-cytoplasmic transporters and NLS-GFP at distances >500 nm from the nuclear envelope. Such a phenomenon would be otherwise invisible at the best resolution of conventional confocal imaging mode. We should stress that, in principle, the photophysics of the fluorescent reporter in STED conditions is the only limiting the resolution in stFCS diffusional maps.[5]1. M. A. Digman and E. Gratton, Annu Rev Phys Chem 62, 645-668 (2011).2. F. Cardarelli, R. Bizzarri, M. Serresi, L. Albertazzi, and F. Beltram, J Biol Chem 284(52), 36638-36646 (2009).3. A. Diaspro, Nanoscopy and Multidimensional Optical Fluorescence Microscopy‎, 448 (Chapman and Hall/CRC, Boca Raton, USA, 2010).4. S. Galiani, B. Harke, G. Vicidomini, G. Lignani, F. Benfenati, A. Diaspro, and P. Bianchini, Opt Express 20(7), 7362-7374 (2012).5. S. W. Hell, Science 316(5828), 1153-1158 (2007).

Synthesis and Application of Visible Light Sensitive Azobenzene

Methods for regulating peptide conformation by non-harmful light stimuli can be useful for remotely controlling cellular functions in vitro. Here, we synthesized a series of p-heteroatom-substituted azobenzenes and studied their photoisomerization properties. The trans-isomer of p-sulfur-substituted azobenzene was effectively isomerized by visible light irradiation and the cis-isomer was thermally stable at physiological temperature. We developed a novel visible light sensitive amino acid (AZO), via p-sulfur-substituted azobenzene, and utilized it as a photosensitive modulator of the SV40 nuclear localization signal (NLS). The cellular uptake of the AZO-NLS conjugate was controlled by visible light irradiation. Our technology can be utilized for regulating not only the cellular uptake, but also the function of peptides within cells by non-harmful visible light irradiation.

DNA damage checkpoint triggers autophagy to regulate the initiation of anaphase

Budding yeast cells suffering a single unrepaired double-strand break (DSB) trigger the Mec1 (ATR)-dependent DNA damage response that causes them to arrest before anaphase for 12-15 h. Here we find that hyperactivation of the cytoplasm-to-vacuole (CVT) autophagy pathway causes the permanent G2/M arrest of cells with a single DSB that is reflected in the nuclear exclusion of both Esp1 and Pds1. Transient relocalization of Pds1 is also seen in wild-type cells lacking vacuolar protease activity after induction of a DSB. Arrest persists even as the DNA damage-dependent phosphorylation of Rad53 diminishes. Permanent arrest can be overcome by blocking autophagy, by deleting the vacuolar protease Prb1, or by driving Esp1 into the nucleus with a SV40 nuclear localization signal. Autophagy in response to DNA damage can be induced in three different ways: by deleting the Golgi-associated retrograde protein complex (GARP), by adding rapamycin, or by overexpression of a dominant ATG13-8SA mutation.

Physicians and Advanced Practice Registered Nurses: Working Together As a Team

The phytohormone abscisic acid (ABA) promotes plant tolerance to major stresses like drought, partly by modulating plant growth and development. However, the underlying mechanisms are poorly understood. Here, we show that cell proliferation in the *Arabidopsis thaliana* root meristem is controlled by the interplay between three kinases, SNF1-RELATED KINASE 2 (SnRK2), the main driver of ABA signaling, the SnRK1 energy sensor, and the growth-promoting TARGET OF RAPAMYCIN (TOR) kinase. Under favorable conditions, the SnRK1α1 catalytic subunit is enriched in the nuclei of root meristematic cells and this is accompanied by normal cell proliferation and meristem size. Depletion of SnRK2s in a *snrk2.2 snrk2.3* double mutant causes constitutive cytoplasmic localization of SnRK1α1 and a reduction in meristem size, suggesting that, under non-stress conditions, SnRK2s enable growth by retaining SnRK1α1 in the nucleus. In response to elevated ABA levels, SnRK1α1 translocates to the cytoplasm and this is accompanied by inhibition of TOR, decreased cell proliferation and meristem size. Blocking nuclear export with leptomycin B abrogates ABA-driven SnRK1α1 relocalization to the cytoplasm and the inhibition of TOR. Fusion of SnRK1α1 to an SV40 nuclear localization signal leads to defective TOR repression in response to ABA, demonstrating that SnRK1α1 nuclear exit is a premise for this repression. Finally, the SnRK2-dependent changes in SnRK1α1 subcellular localization are specific to the proliferation zone of the meristem, underscoring the relevance of this mechanism for growth regulation.

NBA1/MERIT40 and BRE Interaction Is Required for the Integrity of Two Distinct Deubiquitinating Enzyme BRCC36-containing Complexes

BRCC36-deubiquitinating enzyme (DUB) forms two different complexes through interactions with two different adaptor proteins Abraxas and ABRO1 in cells. Abraxas mainly localizes in the nucleus, mediating the interaction of BRCC36 with BRCA1. ABRO1 is mainly localized in the cytoplasm. Because it lacks the BRCA1-interacting motif, the ABRO1 complex does not interact with BRCA1. Both BRCC36-containing complexes contain common components including BRE and NBA1/MERIT40. Here, we found that the two complexes are assembled in a similar manner and NBA1 and BRE interaction is critical for maintaining the integrity of both of the complexes. Knockdown of NBA1 or BRE leads to decreased levels of components of the two BRCC36-containing complexes. We provided evidence that NBA1 interacts with BRE through a C-terminal conserved motif of the NBA1 protein and a C-terminal UEV domain of the BRE protein. Furthermore, the NBA1-BRE interaction is required for cellular resistance to ionizing irradiation and NBA1's role in recruiting BRCA1 to DNA damage sites. Together, these studies reveal critical interactions required for the formation and function of BRCC36-containing DUB complexes.