Biochemistry and Cell Biology

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A gain-of-function mutation in IAA16 confers reduced responses to auxin and abscisic acid and impedes plant growth and fertility
(Springer, 2012) Rinaldi, Mauro A.; Liu, James; Enders, Tara A.; Bartel, Bonnie; Strader, Lucia C.
Auxin regulates many aspects of plant development, in part, through degradation of the Aux/IAA family of transcriptional repressors. Consequently, stabilizing mutations in several Aux/IAA proteins confer reduced auxin responsiveness. However, of the 29 apparent Aux/IAA proteins in Arabidopsis thaliana, fewer than half have roles established through mutant analysis. We identified iaa16-1, a dominant gain-of-function mutation in IAA16 (At3g04730), in a novel screen for reduced root responsiveness to abscisic acid. The iaa16-1 mutation also confers dramatically reduced auxin responses in a variety of assays, markedly restricts growth of adult plants, and abolishes fertility when homozygous. We compared iaa16-1 phenotypes with those of dominant mutants defective in the closely related IAA7/AXR2, IAA14/SLR, and IAA17/AXR3, along with the more distantly related IAA28, and found overlapping but distinct patterns of developmental defects. The identification and characterization of iaa16-1 provides a fuller understanding of the IAA7/IAA14/IAA16/IAA17 clade of Aux/IAA proteins and the diverse roles of these repressors in hormone response and plant development.
A hydrogel-based tumor model for the evaluation of nanoparticle-based cancer therapeutics
(Elsevier, 2014) Xu, Xian; Sabanayagam, Chandran R.; Harrington, Daniel Anton; Farach-Carson, Mary C.; Jia, Xinqiao
Three-dimensional (3D) tissue-engineered tumor models have the potential to bridge the gap between monolayer cultures and patient-derived xenografts for the testing of nanoparticle (NP)-based cancer therapeutics. In this study, a hydrogel-derived prostate cancer (PCa) model was developed for the in vitro evaluation of doxorubicin (Dox)-loaded polymer NPs (Dox-NPs). The hydrogels were synthesized using chemically modified hyaluronic acid (HA) carrying acrylate groups (HA-AC) or reactive thiols (HA-SH). The crosslinked hydrogel networks exhibited an estimated pore size of 70–100 nm, similar to the spacing of the extracellular matrices (ECM) surrounding tumor tissues. LNCaP PCa cells entrapped in the HA matrices formed distinct tumor-like multicellular aggregates with an average diameter of 50 μm after 7 days of culture. Compared to cells grown on two-dimensional (2D) tissue culture plates, cells from the engineered tumoroids expressed significantly higher levels of multidrug resistance (MDR) proteins, including multidrug resistance protein 1 (MRP1) and lung resistance-related protein (LRP), both at the mRNA and the protein levels. Separately, Dox-NPs with an average diameter of 54 ± 1 nm were prepared from amphiphilic block copolymers based on poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) bearing pendant cyclic ketals. Dox-NPs were able to diffuse through the hydrogel matrices, penetrate into the tumoroid and be internalized by LNCaP PCa cells through caveolae-mediated endocytosis and macropinocytosis pathways. Compared to 2D cultures, LNCaP PCa cells cultured as multicellular aggregates in HA hydrogel were more resistant to Dox and Dox-NPs treatments. Moreover, the NP-based Dox formulation could bypass the drug efflux function of MRP1, thereby partially reversing the resistance to free Dox in 3D cultures. Overall, the engineered tumor model has the potential to provide predictable results on the efficacy of NP-based cancer therapeutics.
A novel in vivo model for evaluating functional restoration of a tissue-engineered salivary gland
(Wiley, 2014) Pradhan-Bhatt, Swati; Harrington, Daniel Anton; Duncan, Randall L.; Farach-Carson, Mary C.; Jia, Xinqiao; Witt, Robert L.
Objectives/Hypothesis: To create a novel model for development of a tissue-engineered salivary gland from human salivary gland cells that retains progenitor cell markers useful for treatment of radiation-induced xerostomia. Study Design: A three-dimensional (3D) hyaluronic acid (HA)-based hydrogel scaffold was used to encapsulate primary human salivary gland cells and to obtain organized acini-like spheroids. Hydrogels were implanted into rat models, and cell viability and receptor expression were evaluated. Methods: A parotid gland surgical resection model for xenografting was developed. Salivary cells loaded in HA hydrogels formed spheroids and in vitro were implanted in the three-fourths resected parotid bed of athymic rats. Implants were removed after 1 week and analyzed for spheroid viability and phenotype retention. Results: Spheroids in 3D stained positive for HA receptors CD168/RHAMM and CD44, which is also a progenitor cell marker. The parotid gland three-fourths resection model was well-tolerated by rodent hosts, and the salivary cell/hydrogel scaffolds were adherent to the remaining parotid gland, with no obvious signs of inflammation. A majority of human cells in the extracted hydrogels demonstrated robust expression of CD44. Conclusions: A 3D HA-based hydrogel scaffold that supported long-term culture of salivary gland cells into organized spheroids was established. An in vivo salivary gland resection model was developed that allowed for integration of the 3D HA hydrogel scaffold with the existing glandular parenchyma. The expression of CD44 among salivary cultures may partially explain their regenerative potential, and the expression of CD168/RHAMM along with CD44 may aid the development of these 3D spheroids into regenerated salivary glands.
A promoter-level mammalian expression atlas
(Nature Publishing Group, 2014) FANTOM Consortium; RIKEN PMI; CLST (DGT)
Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly ‘housekeeping’, whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.
A role for the root cap in root branching revealed by the non-auxin probe naxillin
(Nature America, Inc., 2012) De Rybel, Bert; Audenaert, Dominique; Xuan, Wei; Overvoorde, Paul; Strader, Lucia C.; Kepinski, Stefan; Hoye, Rebecca; Brisbois, Ronald; Parizot, Boris; Vanneste, Steffan; Liu, Xing; Gilday, Alison; Graham, Ian A.; Nguyen, Long; Jansen, Leentje; Njo, Maria Fransiska; Inze, Dirk; Bartel, Bonnie; Beeckman, Tom
The acquisition of water and nutrients by plant roots is a fundamental aspect of agriculture and strongly depends on root architecture. Root branching and expansion of the root system is achieved through the development of lateral roots and is to a large extent controlled by the plant hormone auxin. However, the pleiotropic effects of auxin or auxin-like molecules on root systems complicate the study of lateral root development. Here we describe a small-molecule screen in Arabidopsis thaliana that identified naxillin as what is to our knowledge the first non-auxin-like molecule that promotes root branching. By using naxillin as a chemical tool, we identified a new function for root cap-specific conversion of the auxin precursor indole-3-butyric acid into the active auxin indole-3-acetic acid and uncovered the involvement of the root cap in root branching. Delivery of an auxin precursor in peripheral tissues such as the root cap might represent an important mechanism shaping root architecture.
A secreted protein is an endogenous chemorepellant in Dictyostelium discoideum
(National Academy of Sciences, 2012) Phillips, Jonathan E.; Gomer, Richard H.; National Institutes of Health
Chemorepellants may play multiple roles in physiological and pathological processes.However, few endogenous chemorepellants have been identified, and how they function is unclear. We found that the autocrine signal AprA, which is produced by growing Dictyostelium discoideum cells and inhibits their proliferation, also functions as a chemorepellant.Wild-type cells at the edge of a colony show directed movement outward from the colony, whereas cells lacking AprA do not. Cells show directed movement away from a source of recombinant AprA and dialyzed conditioned media from wild-type cells, but not dialyzed conditionedmedia fromaprA− cells. The secreted protein CfaD, the G protein Gα8, and the kinase QkgA are necessary for the chemorepellant activity of AprA as well as its proliferation-inhibiting activity, whereas the putative transcription factor BzpN is dispensable for the chemorepellant activity of AprA but necessary for inhibition of proliferation. Phospholipase C and PI3 kinases 1 and 2, which are necessary for the activity of at least one other chemorepellant in Dictyostelium, are not necessary for recombinant AprA chemorepellant activity. Starved cells are not repelled by recombinant AprA, suggesting that aggregation-phase cells are not sensitive to the chemorepellant effect. Cell tracking indicates that AprA affects the directional bias of cell movement, but not cell velocity or the persistence of cell movement. Together, our data indicate that the endogenous signal AprA acts as an autocrine chemorepellant for Dictyostelium cells.
A Technique to Increase Accessibility to Late-Stage Chick Embryos for In Ovo Manipulations
(Wiley, 2013) Spurlin, James III; Lwigale, Peter Y.
Background: During early development, avian embryos are easily accessible in ovo for transplantations and experimental perturbations. However, these qualities of the avian embryonic model rapidly wane shortly after embryonic day (E)4 when the embryo is obscured by extraembryonic membranes, making it difficult to study developmental events that occur at later stages in vivo. Results: In this study, we describe a multistep method that involves initially windowing eggs at E3, followed by dissecting away extraembryonic membranes at E5 to facilitate embryo accessibility in ovo until later stages of development. The majority of the embryos subjected to this technique remain exposed between E5 and E8, then become gradually displaced by the growing allantois from posterior to anterior regions. Conclusions: Exposed embryos are viable and compatible with embryological and modern developmental biology techniques including tissue grafting and ablation, gene manipulation by electroporation, and protein expression. This technique opens up new avenues for studying complex cellular interactions during organogenesis and can be further extrapolated to regeneration and stem cell studies.
A tRNA splicing operon: Archease endows RtcB with dual GTP/ATP cofactor specificity and accelerates RNA ligation
(Oxford University Press, 2014) Desai, Kevin K.; Cheng, Chin L.; Bingman, Craig A.; Phillips, George N.Jr.; Raines, Ronald T.
Archease is a 16-kDa protein that is conserved in all three domains of life. In diverse bacteria and archaea, the genes encoding Archease and the tRNA ligase RtcB are localized into an operon. Here we provide a rationale for this operon organization by showing that Archease and RtcB from Pyrococcus horikoshii function in tandem, with Archease altering the catalytic properties of the RNA ligase. RtcB catalyzes the GTP and Mn(II)-dependent joining of either 2′,3′-cyclic phosphate or 3′-phosphate termini to 5′-hydroxyl termini. We find that catalytic concentrations of Archease are sufficient to activate RtcB, and that Archease accelerates both the RNA 3′-P guanylylation and ligation steps. In addition, we show that Archease can alter the NTP specificity of RtcB such that ATP, dGTP or ITP is used efficiently. Moreover, RtcB variants that have inactivating substitutions in the guanine-binding pocket can be rescued by the addition of Archease. We also present a 1.4 Å-resolution crystal structure of P. horikoshii Archease that reveals a metal-binding site consisting of conserved carboxylates located at the protein tip. Substitution of the Archease metal-binding residues drastically reduced Archease-dependent activation of RtcB. Thus, evolution has sought to co-express archease and rtcB by creating a tRNA splicing operon.
A viable Arabidopsis pex13 missense allele confers severe peroxisomal defects and decreases PEX5 association with peroxisomes
(Springer, 2014) Woodward, Andrew W.; Fleming, Wendell A.; Burkhart, Sarah E.; Ratzel, Sarah E.; Bjornson, Marta; Bartel, Bonnie
Peroxisomes are organelles that catabolize fatty acids and compartmentalize other oxidative metabolic processes in eukaryotes. Using a forward-genetic screen designed to recover severe peroxisome-defective mutants, we isolated a viable allele of the peroxisome biogenesis gene PEX13 with striking peroxisomal defects. The pex13-4 mutant requires an exogenous source of fixed carbon for pre-photosynthetic development and is resistant to the protoauxin indole-3-butyric acid. Delivery of peroxisome-targeted matrix proteins depends on the PEX5 receptor docking with PEX13 at the peroxisomal membrane, and we found severely reduced import of matrix proteins and less organelle-associated PEX5 in pex13-4 seedlings. Moreover, pex13-4 physiological and molecular defects were partially ameliorated when PEX5 was overexpressed, suggesting that PEX5 docking is partially compromised in this mutant and can be improved by increasing PEX5 levels. Because previously described Arabidopsis pex13 alleles either are lethal or confer only subtle defects, the pex13-4 mutant provides valuable insight into plant peroxisome receptor docking and matrix protein import.
Adaptation of Enterococcus faecalis to Daptomycin Reveals an Ordered Progression to Resistance
(American Society for Microbiology, 2013) Miller, Corwin A.; Kong, Jiayi; Tran, Truc T.; Arias, Cesar A.; Saxer, Gerda; Shamoo, Yousif
With increasing numbers of hospital-acquired antibiotic resistant infections each year and staggering health care costs, there is a clear need for new antimicrobial agents, as well as novel strategies to extend their clinical efficacy. While genomic studies have provided a wealth of information about the alleles associated with adaptation to antibiotics, they do not provide essential information about the relative importance of genomic changes, their order of appearance, or potential epistatic relationships between adaptive changes. Here we used quantitative experimental evolution of a single polymorphic population in continuous culture with whole-genome sequencing and allelic frequency measurements to study daptomycin (DAP) resistance in the vancomycin-resistant clinical pathogen Enterococcus faecalis S613. Importantly, we sustained both planktonic and nonplanktonic (i.e., biofilm) populations in coculture as the concentration of antibiotic was raised, facilitating the development of more ecological complexity than is typically observed in laboratory evolution. Quantitative experimental evolution revealed a clear order and hierarchy of genetic changes leading to resistance, the signaling and metabolic pathways responsible, and the relative importance of these mutations to the evolution of DAP resistance. Despite the relative simplicity of this ex vivo approach compared to the ecological complexity of the human body, we showed that experimental evolution allows for rapid identification of clinically relevant adaptive molecular pathways and new targets for drug design in pathogens.
Alpha-hemoglobin stabilizing protein (AHSP) markedly decreases the redox potential and reactivity of alpha subunits of human HbA with hydrogen peroxide
(The American Society for Biochemistry and Molecular Biology, Inc., 2012) Mollan, Todd L.; Banerjee, Sambuddha; Wu, Gang; Siburt, Claire J.Parker; Tsai, Ah-Lim; Olson, John S.; Weiss, Mitchell J.; Crumbliss, Alvin L.; Alayash, Abdu I.
Background: AHSP modifies redox properties of bound α subunits. Results: Isolated hemoglobin subunits exhibit significantly different redox properties compared to HbA. A significant decrease in the reduction potential of α subunits bound to AHSP results in preferential binding of ferric α. Conclusion: AHSP:α subunit complexes do not participate in ferric-ferryl heme redox cycling. Significance: AHSP binding to α subunits inhibits subunit pseudoperoxidase activity.
Biochar and Microbial Signaling: Production Conditions Determine Effects on Microbial Communication
(American Chemical Society, 2013) Masiello, Caroline A.; Chen, Ye; Gao, Xiaodong; Liu, Shirley; Cheng, Hsiao-Ying; Bennett, Matthew R.; Rudgers, Jennifer A.; Wagner, Daniel S.; Zygourakis, Kyriacos; Silberg, Jonathan J.
Charcoal has a long soil residence time, which has resulted in its production and use as a carbon sequestration technique (biochar). A range of biological effects can be triggered by soil biochar that can positively and negatively influence carbon storage, such as changing the decomposition rate of organic matter and altering plant biomass production. Sorption of cellular signals has been hypothesized to underlie some of these effects, but it remains unknown whether the binding of biochemical signals occurs, and if so, on time scales relevant to microbial growth and communication. We examined biochar sorption of N-3-oxo-dodecanoyl-L-homoserine lactone, an acyl-homoserine lactone (AHL) intercellular signaling molecule used by many gram-negative soil microbes to regulate gene expression. We show that wood biochars disrupt communication within a growing multicellular system that is made up of sender cells that synthesize AHL and receiver cells that express green fluorescent protein in response to an AHL signal. However, biochar inhibition of AHL-mediated cell–cell communication varied, with the biochar prepared at 700 °C (surface area of 301 m2/g) inhibiting cellular communication 10-fold more than an equivalent mass of biochar prepared at 300 °C (surface area of 3 m2/g). These findings provide the first direct evidence that biochars elicit a range of effects on gene expression dependent on intercellular signaling, implicating the method of biochar preparation as a parameter that could be tuned to regulate microbial-dependent soil processes, like nitrogen fixation and pest attack of root crops.
Biochemical and Structural Evidence in Support of a Coherent Model for the Formation of the Double-Helical Influenza A Virus Ribonucleoprotein
(American Society for Microbiology, 2012) Ye, Qiaozhen; Guu, Tom S.Y.; Mata, Dougslas A.; Kuo, Rei-Lin; Smith, Bartram; Krug, Robert M.; Tao, Yizhi Jane
Influenza A virions contain eight ribonucleoproteins (RNPs), each comprised of a negative-strand viral RNA, the viral polymerase, and multiple nucleoproteins (NPs) that coat the viral RNA. NP oligomerization along the viral RNA is mediated largely by a 28-amino-acid tail loop. Influenza viral RNPs, which serve as the templates for viral RNA synthesis in the nuclei of infected cells, are not linear but rather are organized in hairpin-like double-helical structures. Here we present results that strongly support a coherent model for the assembly of the double-helical influenza virus RNP structure. First, we show that NP self-associates much more weakly in the absence of RNA than in its presence, indicating that oligomerization is very limited in the cytoplasm. We also show that once NP has oligomerized, it can dissociate in the absence of bound RNA, but only at a very slow rate, indicating that the NP scaffold remains intact when viral RNA dissociates from NPs to interact with the polymerase during viral RNA synthesis. In addition, we identify a previously unknown NP-NP interface that is likely responsible for organizing the double-helical viral RNP structure. This identification stemmed from our observation that NP lacking the oligomerization tail loop forms monomers and dimers. We determined the crystal structure of this NP dimer, which reveals this new NP-NP interface. Mutation of residues that disrupt this dimer interface does not affect oligomerization of NPs containing the tail loop but does inactivate the ability of NPs containing the tail loop to support viral RNA synthesis in minigenome assays.
Border patrol: Insights into the unique role of perlecan/heparan sulfate proteoglycan 2 at cell and tissue borders
(Elsevier, 2014) Farach-Carson, Mary C.; Warren, Curtis R.; Harrington, Daniel Anton; Carson, Daniel D.
The extracellular matrix proteoglycan (ECM) perlecan, also known as heparan sulfate proteoglycan 2 or HSPG2, is one of the largest (>ﾠ200ﾠnm) and oldest (>ﾠ550ﾠMﾠyears) extracellular matrix molecules. In vertebrates, perlecan's five-domain structure contains numerous independently folding modules with sequence similarities to other ECM proteins, all connected like cars into one long, diverse complex train following a unique N-terminal domain I decorated with three long glycosaminoglycan chains, and an additional glycosaminoglycan attachment site in the C-terminal domain V. In lower invertebrates, perlecan is not typically a proteoglycan, possessing the majority of the core protein modules, but lacking domain I where the attachment sites for glycosaminoglycan chains are located. This suggests that uniting the heparan sulfate binding growth factor functions of domain I and the core protein functions of the rest of the molecule in domains IIﾖV occurred later in evolution for a new functional purpose. In this review, we surveyed several decades of pertinent literature to ask a fundamental question: Why did nature design this protein uniquely as an extraordinarily long multifunctional proteoglycan with a single promoter regulating expression, rather than separating these functions into individual proteins that could be independently regulated? We arrived at the conclusion that the concentration of perlecan at functional borders separating tissues and tissue layers is an ancient key function of the core protein. The addition of the heparan sulfate chains in domain I likely occurred as an additional means of binding the core protein to other ECM proteins in territorial matrices and basement membranes, and as a means to reserve growth factors in an on-site depot to assist with rapid repair of those borders when compromised, such as would occur during wounding. We propose a function for perlecan that extends its role from that of an extracellular scaffold, as we previously suggested, to that of a critical agent for establishing and patrolling tissue borders in complex tissues in metazoans. We also propose that understanding these unique functions of the individual portions of the perlecan molecule can provide new insights and tools for engineering of complex multi-layered tissues including providing the necessary cues for establishing neotissue borders.
Cell-specific transmembrane injection of molecular cargo with gold nanoparticle-generated transient plasmonic nanobubbles
(Elsevier, 2012) Lukianova-Hleb, Ekaterina Y.; Wagner, Daniel S.; Brenner, Malcolm K.; Lapotko, Dmitri O.
Optimal cell therapies require efficient, selective and rapid delivery of molecular cargo into target cells without compromising their viability. Achieving these goals exﾠvivo in bulk heterogeneous multi-cell systems such as human grafts is impeded by low selectivity and speed of cargo delivery and by significant damage to target and non-target cells. We have developed a cell level approach for selective and guided transmembrane injection of extracellular cargo into specific target cells using transient plasmonic nanobubbles (PNB) as cell-specific nano-injectors. As a technical platform for this method we developed a laser flow cell processing system. The PNB injection method and flow system were tested in heterogeneous cell suspensions of target and non-target cells for delivery of Dextran-FITC dye into squamous cell carcinoma HN31 cells and transfection of human T-cells with a green fluorescent protein-encoding plasmid. In both models the method demonstrated single cell type selectivity, high efficacy of delivery (96% both for HN31 cells T-cells), speed of delivery (nanoseconds) and viability of treated target cells (96% for HN31 cells and 75% for T-cells). The PNB injection method may therefore be beneficial for real time processing of human grafts without removal of physiologically important cells.
Ceruloplasmin Is a Novel Adipokine Which Is Overexpressed in Adipose Tissue of Obese Subjects and in Obesity-Associated Cancer Cells
(Public Library of Science, 2014) Arner, Erik; Forrest, Alistair R.R.; Ehrlund, Anna; Mejhert, Niklas; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Laurencikiene, Jurga; Rydén, Mikael; Arner, Peter; FANTOM Consortium
Obesity confers an increased risk of developing specific cancer forms. Although the mechanisms are unclear, increased fat cell secretion of specific proteins (adipokines) may promote/facilitate development of malignant tumors in obesity via cross-talk between adipose tissue(s) and the tissues prone to develop cancer among obese. We searched for novel adipokines that were overexpressed in adipose tissue of obese subjects as well as in tumor cells derived from cancers commonly associated with obesity. For this purpose expression data from human adipose tissue of obese and non-obese as well as from a large panel of human cancer cell lines and corresponding primary cells and tissues were explored. We found expression of ceruloplasmin to be the most enriched in obesity-associated cancer cells. This gene was also significantly up-regulated in adipose tissue of obese subjects. Ceruloplasmin is the body's main copper carrier and is involved in angiogenesis. We demonstrate that ceruloplasmin is a novel adipokine, which is produced and secreted at increased rates in obesity. In the obese state, adipose tissue contributed markedly (up to 22%) to the total circulating protein level. In summary, we have through bioinformatic screening identified ceruloplasmin as a novel adipokine with increased expression in adipose tissue of obese subjects as well as in cells from obesity-associated cancers. Whether there is a causal relationship between adipose overexpression of ceruloplasmin and cancer development in obesity cannot be answered by these cross-sectional comparisons.
Characterization of the Interaction between the Cohesin Subunits Rad21 and SA1/2
(Public Library of Science, 2013) Zhang, Nenggang; Jiang, Yunyun; Mao, Qilong; Demeler, Borries; Tao, Yizhi Jane; Pati, Debananda
The cohesin complex is responsible for the fidelity of chromosomal segregation during mitosis. It consists of four core subunits, namely Rad21/Mcd1/Scc1, Smc1, Smc3, and one of the yeast Scc3 orthologs SA1 or SA2. Sister chromatid cohesion is generated during DNA replication and maintained until the onset of anaphase. Among the many proposed models of the cohesin complex, the ﾒcoreﾒ cohesin subunits Smc1, Smc3, and Rad21 are almost universally displayed as tripartite ring. However, other than its supportive role in the cohesin ring, little is known about the fourth core subunit SA1/SA2. To gain deeper insight into the function of SA1/SA2 in the cohesin complex, we have mapped the interactive regions of SA2 and Rad21 in vitro and ex vivo. Whereas SA2 interacts with Rad21 through a broad region (301ﾖ750 aa), Rad21 binds to SA proteins through two SA-binding motifs on Rad21, namely N-terminal (NT) and middle part (MP) SA-binding motif, located At 60-81 aa of the N-terminus and 383ﾖ392 aa of the MP of Rad21, respectively. The MP SA-binding motif is a 10 amino acid, a-helical motif. Deletion of these 10 amino acids or mutation of three conserved amino acids (L385, F389, and T390) in this ahelical motif significantly hinders Rad21 from physically interacting with SA1/2. Besides the MP SA-binding motif, the NT SAbinding motif is also important for SA1/2 interaction. Although mutations on both SA-binding motifs disrupt Rad21-SA1/2 interaction, they had no apparent effect on the Smc1-Smc3-Rad21 interaction. However, the Rad21-Rad21 dimerization was reduced by the mutations, indicating potential involvement of the two SA-binding motifs in the formation of the two-ring handcuff for chromosomal cohesion. Furthermore, mutant Rad21 proteins failed to significantly rescue precocious chromosome separation caused by depletion of endogenous Rad21 in mitotic cells, further indicating the physiological significance of the two SA-binding motifs of Rad21.
CIRCADIAN CLOCK-ASSOCIATED1 Controls Resistance to Aphids by Altering Indole Glucosinolate Production
(Oxford University Press, 2019) Lei, Jiaxin; Jayaprakasha, Guddadarangavvanahally K.; Singh, Jashbir; Uckoo, Rammohan; Borrego, Eli J.; Finlayson, Scott; Kolomiets, Mike; Patil, Bhimanagouda S.; Braam, Janet; Zhu-Salzman, Keyan
CIRCADIAN CLOCK-ASSOCIATED1 (CCA1), a well-known central circadian clock regulator, coordinates plant responses to environmental challenges. Its daily rhythmic expression in Arabidopsis (Arabidopsis thaliana) confers host resistance to the caterpillar Trichoplusia ni. However, it is unclear whether CCA1 plays a role in defense against phloem sap-feeding aphids. In this study, we showed that green peach aphid (Myzus persicae) displayed an intrinsic circadian feeding rhythm. Under constant light, wild-type Columbia-0 (Col-0) Arabidopsis plants coentrained with aphids in the same light/dark cycles exhibited greater antixenotic activity than plants preentrained in the opposite cycle from the aphids. Consistently, circadian mutants cca1-1, cca1-11, lhy-21, ztl-1, ztl-4, and lux-2 suffered more severe damage than Col-0 plants when infested by aphids, suggesting that the Arabidopsis circadian clock plays a defensive role. However, the arrhythmic CCA1 overexpression line (CCA1-OX) displayed strong antixenotic and antibiotic activities despite its loss of circadian regulation. Aphids feeding on CCA1-OX plants exhibited lower reproduction and smaller body size and weight than those on Col-0. Apparently, CCA1 regulates both clock-dependent and -independent defense responses. Systematic investigation based on bioinformatics analyses indicated that resistance to aphids in CCA1-OX plants was due primarily to heightened basal indole glucosinolate levels. Interestingly, aphid feeding induced alternatively spliced intron-retaining CCA1a/b transcripts, which are normally expressed at low levels, whereas expression of the major fully spliced CCA1 transcript remained largely unchanged. We hypothesize that posttranscriptional modulation of CCA1 expression upon aphid infestation maximizes the potential of circadian-mediated defense and stress tolerance while ensuring normal plant development.
Circadian control of jasmonates and salicylates: The clock role in plant defense
(Landes Bioscience, 2013) Goodspeed, Danielle; Chehab, E.Wassim; Covington, Michael F.; Braam, Janet
Plants have evolved robust mechanisms to perceive and respond to diverse environmental stimuli.ﾠ The plant phytohormones jasmonates and salicylates play key roles in activating biotic stress response pathways. Recent findings demonstrate that basal levels of both jasmonates and salicylates in Arabidopsis are under the control of the circadian clock and that clock-controlled jasmonate accumulation may underlie clock- and jasmonate-dependent enhanced resistance of Arabidopsis to Trichoplusia ni (cabbage looper), a generalist herbivore. Here we summarize these findings and provide further evidence that a functional plant circadian clock is required for optimal herbivore defense in Arabidopsis.ﾠ When given a choice to feed on wild-type plants or arrhythmic transgenics, T. ni prefer plants lacking robust circadian rhythms. Altogether these data provide strong evidence for circadian clock enabling anticipation of herbivore attack and thus contributing to overall plant fitness.
CLN8 is an endoplasmic reticulum cargo receptor that regulates lysosome biogenesis
(Springer Nature, 2018) di Ronza, Alberto; Bajaj, Lakshya; Sharma, Jaiprakash; Sanagasetti, Deepthi; Lotfi, Parisa; Adamski, Carolyn Joy; Collette, John; Palmieri, Michela; Amawi, Abdallah; Popp, Lauren; Chang, Kevin Tommy; Meschini, Maria Chiara; Leung, Hon-Chiu Eastwood; Segatori, Laura; Simonati, Alessandro; Sifers, Richard Norman; Santorelli, Filippo Maria; Sardiello, Marco
Organelle biogenesis requires proper transport of proteins from their site of synthesis to their target subcellular compartment1,2,3. Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and traffic through the Golgi complex before being transferred to the endolysosomal system4,5,6, but how they are transferred from the ER to the Golgi is unknown. Here, we show that ER-to-Golgi transfer of lysosomal enzymes requires CLN8, an ER-associated membrane protein whose loss of function leads to the lysosomal storage disorder, neuronal ceroid lipofuscinosis 8 (a type of Batten disease)7. ER-to-Golgi trafficking of CLN8 requires interaction with the COPII and COPI machineries via specific export and retrieval signals localized in the cytosolic carboxy terminus of CLN8. CLN8 deficiency leads to depletion of soluble enzymes in the lysosome, thus impairing lysosome biogenesis. Binding to lysosomal enzymes requires the second luminal loop of CLN8 and is abolished by some disease-causing mutations within this region. Our data establish an unanticipated example of an ER receptor serving the biogenesis of an organelle and indicate that impaired transport of lysosomal enzymes underlies Batten disease caused by mutations in CLN8.

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