This data strongly suggest that sumoylation of mouse PML can be faithfully reproduced in yeast by employing its endogenous sumoylation machinery. In line with previous observations  overexpression of PML resulted in an overall increase of Smt3-conjugated species, most likely representing Smt3 linked to PML and to endogenous yeast proteins Fig. How PML stimulates Smt3 conjugation in yeast remains to be determined. In conclusion, we show that the use of a model eukaryote, in combination with a highly versatile plasmid system that allows regulated expression of multiple heterologous proteins, can facilitate the analysis of complex mammalian post-translational modification cascades such as ubiquitination and sumoylation in a cellular context.
A complete list of plasmids generated in this study can be found in Table S1. Plasmids will be deposited at Addgene. Strain yRA1 was derived from S. This deletion keeps elements of the UAS upstream activating sequence required for expression of the adjacent GAL3 gene intact thereby not affecting the kinetics of galactose induction . Transformation of S. At the desired time points, samples were taken and the absorbance measured at nm. Samples were taken at the indicated time points and total protein lysates were prepared. Samples were collected at the indicated time points in ice-cold sodium azide 10 mM final concentration to prevent undesired protein degradation.
Protein extracts were prepared by breaking the cells with glass beads and acid precipitation essentially as previously described . Aceton washed protein pellets were dried, re-suspended in 1X Laemmli-sample buffer 0. Protein transfer and detection was done essentially as previously described . Download Supplemental Information. Research Articles: Microbial Cell, Vol. Abstract Ubiquitination is a posttranslational protein modification that regulates most aspects of cellular life. Degradation of E6-E7 To test if E6AP-dependent poly-ubiquitination of E6-E7 leads to proteasome-mediated degradation of the fusion protein in the reconstituted yeast system, we performed a cycloheximide CHX chase experiment.
Sumoylation of promyelocytic leukemia protein Another example of a biological process that is conserved between mammals and yeast is sumoylation, a post-translational modification very similar to ubiquitination . References A. Swatek, and D. Komander, "Ubiquitin modifications", Cell Research , vol. Metzger, V. Hristova, and A. Buetow, and D. Erpapazoglou, O. Walker, and R. Yau, and M. Rape, "The increasing complexity of the ubiquitin code", Nature Cell Biology , vol. Glickman, and A. Jastrab, and K. Pearce, J. Mintseris, J.
Ferreyra, S. Gygi, and K. Markson, C.
Volatility of Mutator Phenotypes at Single Cell Resolution
Kiel, R. Hyde, S. Brown, P. Charalabous, A. Bremm, J. Semple, J. Woodsmith, S. Duley, K. Salehi-Ashtiani, M. Vidal, D. Komander, L. Serrano, P. Lehner, and C. Sanderson, "Analysis of the human E2 ubiquitin conjugating enzyme protein interaction network", Genome Research , vol. Li, M. Bengtson, A. Ulbrich, A. Matsuda, V. Reddy, A. Orth, S. Chanda, S. Batalov, and C. Keren-Kaplan, I. Attali, K.
Motamedchaboki, B. Davis, N. Tanner, Y. Reshef, E. Laudon, M. Kolot, O. Levin-Kravets, O. Kleifeld, M. Glickman, B. Horazdovsky, D. Wolf, and G. Collins, T. Gomez, R. Deshaies, and W. Tansey, "Combined chemical and genetic approach to inhibit proteolysis by the proteasome", Yeast , vol. Kishino, M. Calculated with Jpred Cole et al. Previously identified conserved regions Milligan et al. Bar graphs and error bars in panels A, B, and D are the result of triplicate experiments with error bars indicating plus or minus one standard deviation. We next assayed Mpp6 effects on degradation of single-stranded polyA RNA, a model substrate for the nuclear exosome, to determine if Mpp6 altered activities of the exosome Liu et al.
Mpp6 stimulated Rrp6 activity by 2. However, Mpp6 could stimulate Dis3 exoribonuclease activity by 3. Mpp6 had no detectable effect on Dis3 endoribonuclease activity Figure 1—figure supplement 1E. Taken together, these results suggest that Mpp6 association with Exo9 can stimulate Rrp6 activity, although stimulation of Dis3 can also be observed in the absence of Rrp6 or in the presence of catalytically inactivated Rrp6.
Mpp6 contains two small regions of high sequence conservation as noted previously Milligan et al. To determine if Rrp6 activity remained dependent on RNA contacts to the Exo9 central channel in the presence of Mpp6, a route of ingress to Rrp6 observed biochemically and structurally in the absence of this nuclear cofactor Wasmuth et al. Data presented in Figure 1 and Figure 1—figure supplement 1 support a model in which Mpp6 binds the Exo9 core and stimulates the activities of the nuclear exosome, most likely through its ability to bind RNA.
While stimulation of Dis3 by Mpp6 was observed in cases where Rrp6 was absent or catalytically inert, the fold stimulation of polyA RNA decay by Rrp6 is among the more striking biochemical effects observed, and is perhaps consistent with the proposed role of Rrp6 in deadenylation of nuclear transcripts in S.
View from side left and top right. B Mpp6 Minimal green makes extensive contacts to Rrp40 orange and spans all three of its domains. C Mpp6 Minimal transparent purple surface in middle and right panels binds to a conserved surface of Rrp Other conserved surfaces important for RNA binding and scaffolding interactions to other exosome subunits are indicated. Surface conservation calculated with ConSurf Ashkenazy et al. Electron densities for Mpp6 residues 90 to were evident on the surface of Rrp40 Figure 2—figure supplement 1A.
Mpp6 Minimal residues 90—97 adopt a distorted parallel beta-strand configuration that complements the second beta-strand of the Rrp40 N-terminal domain NTD while residues 98— adopt a helical conformation that wedges between the Rrp40 NTD and S1 domain Figure 2B. Mpp6 residues — adopt an anti-parallel beta-strand conformation that complements the second beta-strand of the KH domain Figure 2B , Figure 2—figure supplement 1A. Although modeled, densities for amino acids — were very weak in comparison to other portions of Mpp6. Contacts observed in our structure are also consistent with crosslinking and mass spectrometry data reported previously, including crosslinks between Rrp40 Lys and Mpp6 Lys and between Rrp40 Lys49 and Mpp6 Lys Shi et al.
Consistent with the structural data, exosomes lacking Rrp40 could not associate with Mpp6, and Mpp6 did not stimulate Rrp6 activity in exosomes lacking Rrp40 Figure 2—figure supplement 1B,C. Although Mpp6 interacts directly with Rrp40 in the context of the exosome complex, interactions could not be detected between Rrp40 and Mpp6 in isolation Figure 2—figure supplement 1D.
Furthermore, Mpp6 residues 81 to 90, which are disordered in our structure, are required for exosome Rrp6 stimulation Figure 1C. The hydrophobic quality of these Mpp6 residues is conserved within eukaryotes, despite divergent sequence Figure 3A. While Mpp6 residues — are not conserved across evolution, Tyr99 is conserved as tyrosine, phenylalanine or leucine in other Mpp6 family members. Additional hydrophobic interactions are observed within the last beta strand of Mpp6, including contacts between Mpp6 Phe and Rrp40 Phe Figure 3B.
In yeast through man, the corresponding residue in Mpp6 is either a phenylalanine or tryptophan Figure 3A , while a phenylalanine or leucine is present in Rrp Bottom: sequence alignment among S. Sequences for S. Sequence alignment and conservation calculated with Clustal Omega Larkin et al.
Three regions a, b, c of high conservation are noted. B Stick representation of conserved contacts between Mpp6 green and Rrp40 orange are noted. C Mpp6 arginine residues were individually substituted to glutamate residues to interrogate the importance of the arginine anchor. Mutations disrupting the arginine anchor, Arg, are most detrimental to Mpp6-mediated stimulation of Rrp6 activity.
An additive effect is observed when a distal Mpp6 mutation, Arg89, is combined with a mutation in Rrp40 EA that disrupts coordination of the Mpp6 arginine anchor. The most prominent electron density observed for Mpp6 corresponds to Arg within the last beta strand. Arg projects into a pocket formed between the S1 and KH domains of Rrp40 Figure 3B ; Figure 2—figure supplement 1A and resides in one of two regions of Mpp6 previously identified as conserved from yeast to man Milligan et al. Arg is within hydrogen bond distance to the Glu side chain carboxylate and the backbone carbonyl oxygen of Gly Figure 3B , residues that are not known to be involved in other interactions and are strictly conserved in Rrp Interestingly, two known mutations within Rrp40 that are associated with neurological disorders lie proximal to the arginine anchor of Mpp6.
These include Gly Gly in human , which is mutated to cysteine in hereditary spastic paraplegia in humans Halevy et al. It is unclear how these mutations exert their phenotype or if they result in a loss of interaction with Mpp6. RE exhibited the greatest defect, as would be predicted from removal of the arginine anchor Figure 3C. The defect on Exo10 Rrp6 stimulation observed for a second mutant, Mpp6 R89E, was exacerbated when combined with Rrp40 EA, a mutation predicted to disrupt contacts to the Mpp6 arginine anchor Figure 3B.
The third Mpp6 mutant, RE, exhibited a weaker defect that could be overcome by adding fold excess mutant Mpp6. In contrast, the defect in exosome stimulation by Mpp6 RE was not overcome by addition of excess cofactor Figure 3—figure supplement 1. These results are consistent with contacts observed in the structure and suggest that Mpp6 association is required for stimulation of Rrp6 in the nuclear RNA exosome.
Unassigned electron densities were located between Mpp6, Rrp43 and Rrp6. Anomalous peaks suggest that most unassigned densities likely correspond to a disordered loop in Rrp43 as Rrp43 Met is located proximal to this region Figure 4—figure supplement 1A. Rrp6, Rrp43, Mpp6 and Rrp40 shown in cartoon and stick representation under a transparent surface.
B Schematic domain structure of Rrp6. C Exo10 Rrp6 lacking part or all of the Rrp6 lasso are less stimulated by Mpp6 than Exo10 Rrp6 containing an intact lasso Bar graphs and error bars in panels C , D , and E are the result of triplicate experiments with error bars indicating plus or minus one standard deviation. To determine if the Rrp6 lasso contributes to Mpp6-mediated stimulation of exosome activity, Exo10 Rrp6 was reconstituted with an intact lasso , partial lasso or lassoless Rrp6 and assayed for decay of polyA RNA in the presence or absence of Mpp6 Figure 4C.
The Rrp6 lasso was necessary for Mpp6-dependent stimulation of exosome activity, regardless of whether the lasso was intact residues — or composed of its distal half residues — Figure 4—figure supplement 1C. Collectively, these results suggest that Mpp6 cooperates with the Rrp6 lasso to stimulate the nuclear exosome.
Biochemical results thus far suggest that Mpp6 can stimulate the nuclear exosome. Previous biochemical studies have not revealed Rrpmediated stimulation of Rrp6 activity, but these studies used either truncated Rrp47 Schuch et al. To biochemically characterize the effects of Mpp6 and Rrp47 on nuclear exosome-mediated RNA decay, we repeated assays using full-length proteins given the recent reported relevance of the Rrp6 lasso Wasmuth and Lima, Initial rates from triplicate experiments shown, with error bars representing plus or minus one standard deviation.
Initial rates from triplicate experiments are shown, with error bars representing plus or minus one standard deviation. Bar graphs represent the ratio of Mtr4 kDa to Dis3 kDa in peak fractions of the complex Figure 5—figure supplement 1C , as calculated by densitometric analysis of the fractions on SDS-PAGE, with error bars representing plus or minus one standard deviation. This observation suggests that the stimulatory effects of Mpp6 and Rrp47 depend on unique elements within the nuclear exosome. These biochemical observations appear consistent with recent observations in yeast wherein deletion of the Rrp6 PMC2NT domain, a mutation known to result in loss of Rrp47 interaction Stead et al.
How might Mpp6 and Rrp47 stimulate Rrp6 activity in the nuclear exosome? It remains unclear if Mpp6 and Rrp47 are responsible for the observed differences in Rrp6 conformations. Results presented thus far do not explain the synthetic lethality observed when Mpp6 is deleted along with Rrp6, or Air1, a component of the TRAMP complex Milligan et al. However, synthetic lethality was observed when mutations in the tripartite interaction with Mtr4 were combined with a Mtr4 C-terminal GFP tag, consistent with Mtr4 recruitment to the exosome being dependent on additional factors or Mtr4 surfaces that interact with Rrp6, Mpp6, Rrp47 or other components of the TRAMP complex.
Furthermore, experiments in human suggest that both Mpp6 and Rrp47 contribute to Mtr4 recruitment Chen et al. Consistent with previous results, Mtr4 did not co-elute with the exosome in the absence of Rrp47 and Mpp6 while co-elution was observed in the presence of Rrp47 Schuch et al. When Mpp6 and Rrp47 were both present, more Mtr4 was detected in fractions containing the exosome suggesting greater stability of the complex.
Importantly, Mpp6 was capable of recruiting Mtr4 to the exosome in the absence of Rrp47 and, to lesser extent, in the absence of Rrp6. Removing the C-terminal 30 amino acids of Mpp6 did not disrupt Mtr4 interaction, but removal of the N-terminal 81 amino acids in Mpp6 or the conserved N-terminal 22 amino acid motif in Mpp6 resulted in no detectable interactions with Mtr4. Mpp6 Minimal failed to interact with Mtr4.
These data suggest that Mpp6 elements required for exosome activation are distinct from those required for Mtr4 recruitment. Previous studies reported that Dis3 is unable to degrade the ds 17 A 10 substrate in the nuclear exosome Makino et al. Products of Dis3 activity could be detected with the ds 17 A 10 substrate, but only when Rrp47 or Mpp6 was present.
As before, Mtr4-dependent decay was only observed in the presence of Rrp47 or Mpp6, while addition of both cofactors resulted in the greatest stimulation Figure 6A. These results show that Mtr4 can promote degradation of dsRNA by the nuclear exosome, and that this activity is dependent on Mpp6 and Rrp47 cofactors.
Samples were not heated prior to gel electrophoresis, so the dsRNA substrate runs as a duplex at approximately 37 nt A Activity of reconstituted RNA exosome complexes in the presence or absence of equimolar exogenous Mtr4. In silico density functional theory calculations suggested graviquinone as a kinetic product of pcm-scavenging OH -O-center dot radicals. Our results demonstrate the pharmacological value of an in situ-formed, oxidative stress-related metabolite of an antioxidant.
This might be of particular importance for designing new strategies for antioxidant-based drug discovery. Adaptive immune response is part of the dynamic changes that accompany motoneuron loss in amyotrophic lateral sclerosis ALS. Our results suggest that self-directed immune response takes place during the course of the disease, contributing to the selective elimination of a subset of motoneurons in ALS. Biomarkers with high reproducibility and accurate prediction performance can contribute to comprehending the underlying pathogenesis of related complex diseases and further facilitate disease diagnosis and therapy.
Techniques integrating gene expression profiles and biological networks for the identification of network-based disease biomarkers are receiving increasing interest. The biomarkers for heterogeneous diseases often exhibit strong cooperative effects, which implies that a set of genes may achieve more accurate outcome prediction than any single gene. In this study, we evaluated various biomarker identification methods that consider gene cooperative effects implicitly or explicitly, and proposed the gene cooperation network to explicitly model the cooperative effects of gene combinations.
The gene cooperation network-enhanced method, named as MarkRank, achieves superior performance compared with traditional biomarker identification methods in both simulation studies and real data sets. The biomarkers identified by MarkRank not only have a better prediction accuracy but also have stronger topological relationships in the biological network and exhibit high specificity associated with the related diseases.
Furthermore, the top genes identified by MarkRank involve crucial biological processes of related diseases and give a good prioritization for known disease genes. In conclusion, MarkRank suggests that explicit modeling of gene cooperative effects can greatly improve biomarker identification for complex diseases, especially for diseases with high heterogeneity.
Surprisingly, given its significance in industrial and medical applications, the structure of calcium D-gluconate has not previously been reported. Unexpectedly, the gluconate crystal structure comprises coordination polymers. Unusually, the calcium coordination number is nine. One of the gluconate ligands contradicts a suggestion from that a straight chain conformation is associated with an intramolecular hydrogen bond. This ligand binds to three adjacent metal centres. Cpf1s, the RNA-guided nucleases of the class II clustered regularly interspaced short palindromic repeats system require a short motive called protospacer adjacent motif PAM to be present next to the targeted sequence for their activity.
Here, we show that two other Cpf1 nucleases, Fn- and MbCpf1, which have been reported to utilize a shorter, more frequently occurring PAM sequence TTN when tested in vitro, carry out efficient genome modification in mammalian cells. Our approach also revealed that besides their activities their PAM preferences are also target dependent.
These variants may become versatile substitutes for wild-type Cpf1s by providing an expanded range of targets for genome engineering applications. Closely related microorganisms often cooperate, but the prevalence and stability of cooperation between different genotypes remain debatable.
Why sign up for an account?
Here, we track the evolution of pellicle biofilms formed through genetic division of labour and ask whether partially deficient partners can evolve autonomy. Pellicles of Bacillus subtilis rely on an extracellular matrix composed of exo-polysaccharide EPS and the fibre protein TasA. In monocultures, Delta eps and Delta tasA mutants fail to form pellicles, but, facilitated by cooperation, they succeed in co-culture.
Interestingly, cooperation collapses on an evolutionary timescale and Delta tasA gradually outcompetes its partner Delta eps. Pellicle formation can evolve independently from division of labour in Delta eps and Delta tasA monocultures, by selection acting on the residual matrix component, TasA or EPS, respectively. Using a set of interdisciplinary tools, we unravel that the TasA producer Delta eps evolves via an unconventional but reproducible substitution in TasA that modulates the biochemical properties of the protein.
Conversely, the EPS producer Delta tasA undergoes genetically variable adaptations, all leading to enhanced EPS secretion and biofilms with different biomechanical properties. Finally, we revisit the collapse of division of labour between Delta eps and Delta tasA in light of a strong frequency versus exploitability trade-off that manifested in the solitarily evolving partners. We propose that such trade-off differences may represent an additional barrier to evolution of division of labour between genetically distinct microorganisms.
We addressed this question by analyzing proteins associated with replication forks in cells with or without FANCJ. To any energy flow there is an associated flow of momentum, so that recoil forces arise every time an object absorbs or deflects incoming energy. This same principle governs the operation of macroscopic turbines as well as that of microscopic turbines that use light as the working fluid.
However, a controlled and precise redistribution of optical energy is not easy to achieve at the micron scale resulting in a low efficiency of power to torque conversion. Here we use direct laser writing to fabricate 3D light guiding structures, shaped as a garden sprinkler, that can precisely reroute input optical power into multiple output channels. The shape parameters are derived from a detailed theoretical analysis of losses in curved microfibers. These optical reaction micro-turbines can maximally exploit light's momentum to generate a strong, uniform and controllable torque.
The compound eye of the fruit fly Drosophila melanogaster is one of the most intensively studied and best understood model organs in the field of developmental genetics. Herein we demonstrate that autophagy, an evolutionarily conserved selfdegradation process of eukaryotic cells, is essential for eye development in this organism.
Autophagic structures accumulate in a specific pattern in the developing eye disc, predominantly in the morphogenetic furrow MF and differentiation zone. Silencing of several autophagy genes Atg in the eye primordium severely affects the morphology of the adult eye through triggering ectopic cell death. In Atg mutant genetic backgrounds however genetic compensatory mechanisms largely rescue autophagic activity in, and thereby normal morphogenesis of, this organ. We also show that in the eye disc the expression of a key autophagy gene, Atg8a, is controlled in a complex manner by the anterior Hox paralog lab labial , a master regulator of early development.
Atg8a transcription is repressed in front of, while activated along, the MF by lab. The amount of autophagic structures then remains elevated behind the moving MF. These results indicate that eye development in Drosophila depends on the cell death-suppressing and differentiating effects of the autophagic process. This novel, developmentally regulated function of autophagy in the morphogenesis of the compound eye may shed light on a more fundamental role for cellular self-digestion in differentiation and organ formation than previously thought.
We describe convergent evidence from transcriptomics, morphology, and physiology for a specialized GABAergic neuron subtype in human cortex. Using unbiased single-nucleus RNA sequencing, we identify ten GABAergic interneuron subtypes with combinatorial gene signatures in human cortical layer 1 and characterize a group of human interneurons with anatomical features never described in rodents, having large 'rosehip'-like axonal boutons and compact arborization. Rosehip cells in layer 1 make homotypic gap junctions, predominantly target apical dendritic shafts of layer 3 pyramidal neurons, and inhibit backpropagating pyramidal action potentials in microdomains of the dendritic tuft.
These cells are therefore positioned for potent local control of distal dendritic computation in cortical pyramidal neurons. X-ray free-electron lasers XFELs enable novel experiments because of their high peak brilliance and femtosecond pulse duration. However, non-superconducting XFELs offer repetition rates of only Hz, placing significant demands on beam time and sample consumption. Given the short spacing between pulses, damage caused by shock waves launched by one XFEL pulse on sample probed by subsequent pulses is a concern. To investigate this issue, we collected data from lysozyme microcrystals, exposed to a similar to 15 mu m XFEL beam.
Under these conditions, data quality is independent of whether the first or subsequent pulses of the train were used for data collection. We also analyzed a mixture of microcrystals of jack bean proteins, from which the structure of native, magnesium-containing concanavalin A was determined. The recruitment and evolutionary optimization of promiscuous enzymes is key to the rapid adaptation of organisms to changing environments.
Our understanding of the precise mechanisms underlying enzyme repurposing is, however, limited: What are the active-site features that enable the molecular recognition of multiple substrates with contrasting catalytic requirements? To gain insights into the molecular determinants of adaptation in promiscuous enzymes, we performed the laboratory evolution of an arylsulfatase to improve its initially weak phenylphosphonate hydrolase activity.
The evolutionary trajectory led to a ,fold enhancement of phenylphosphonate hydrolysis, while the native sulfate and promiscuous phosphate mono-and diester hydrolyses were only marginally affected One of the first events to occur upon DNA damage is the local opening of the compact chromatin architecture, facilitating access of repair proteins to DNA lesions. However, its role in chromatin relaxation at the site of DNA damage has not been explored.
Antibiotic development is frequently plagued by the rapid emergence of drug resistance. However, assessing the risk of resistance development in the preclinical stage is difficult. Standard laboratory evolution approaches explore only a small fraction of the sequence space and fail to identify exceedingly rare resistance mutations and combinations thereof. Therefore, new rapid and exhaustive methods are needed to accurately assess the potential of resistance evolution and uncover the underlying mutational mechanisms.
Here, we introduce directed evolution with random genomic mutations DIvERGE , a method that allows an up to million-fold increase in mutation rate along the full lengths of multiple predefined loci in a range of bacterial species. In a single day, DIvERGE generated specific mutation combinations, yielding clinically significant resistance against trimethoprim and ciprofloxacin. Many of these mutations have remained previously undetected or provide resistance in a species-specific manner. These results indicate pathogen-specific resistance mechanisms and the necessity of future narrow-spectrum antibacterial treatments.
In contrast to prior claims, we detected the rapid emergence of resistance against gepotidacin, a novel antibiotic currently in clinical trials. Based on these properties, DIvERGE could be applicable to identify less resistance-prone antibiotics at an early stage of drug development. Despite the potential obstacle represented by the blood-brain barrier for extravasating malignant cells, metastases are more frequent than primary tumors in the central nervous system.
Not only tightly interconnected endothelial cells can hinder metastasis formation, other cells of the brain microenvironment like astrocytes and microglia can also be very hostile, destroying the large majority of metastatic cells. However, malignant cells that are able to overcome these harmful mechanisms may benefit from the shielding and even support provided by cerebral endothelial cells, astrocytes and microglia, rendering the brain a sanctuary site against anti-tumor strategies.
Thus, cells of the neurovascular unit have a Janus-faced attitude towards brain metastatic cells, being both destructive and protective. In this review, we present the main mechanisms of brain metastasis formation, including those involved in extravasation through the brain vasculature and survival in the cerebral environment. Cerebral pericytes are mural cells embedded in the basement membrane of capillaries.
Increasing evidence suggests that they play important role in controlling neurovascular functions, i. These cells can also influence neuroinflammation which is highly regulated by the innate immune system. Therefore, we systematically tested the pattern recognition receptor expression of brain pericytes. Activation of selected pattern recognition receptors can lead to inflammasome assembly and caspase-dependent secretion of IL-1beta. On the other hand, we could demonstrate secretion of active IL-1beta in response to non-canonical inflammasome activation, i. Our in vitro results indicate that pericytes might have an important regulatory role in neuroinflammation.
Although a multiple feedback loop circuit has been shown to generate the hr rhythm, it remains unclear how robust the clock is in individual cells, or how clock timing is coordinated across the plant. Here we examine clock activity at the single cell level across Arabidopsis seedlings over several days under constant environmental conditions. Our data reveal robust single cell oscillations, albeit desynchronised. In particular, we observe two waves of clock activity; one going down, and one up the root.
We also find evidence of cell-to-cell coupling of the clock, especially in the root tip. A simple model shows that cell-to-cell coupling and our measured period differences between cells can generate the observed waves. Our results reveal the spatial structure of the plant clock and suggest that unlike the centralised mammalian clock, the Arabidopsis clock has multiple coordination points.
We find that loss of Ykt6 leads to large-scale accumulation of autophagosomes that are unable to fuse with lysosomes to form autolysosomes. Of note, loss of Syx5, the partner of Ykt6 in ER-Golgi trafficking does not prevent autolysosome formation, pointing to a more direct role of Ykt6 in fusion.
Interestingly, Ykt6 can be outcompeted from this SNARE complex by Vamp7, and we demonstrate that overexpression of Vamp7 rescues the fusion defect of ykt6 loss of function cells.
- Hypertension in Diabetes.
- Study of Amyloids Using Yeast.
Finally, a point mutant form with an RQ amino acid change in the zero ionic layer of Ykt6 protein that is thought to be important for fusion-competent SNARE complex assembly retains normal autophagic activity and restores full viability in mutant animals, unlike palmitoylation or farnesylation site mutant Ykt6 forms.
As Ykt6 and Vamp7 are both required for autophagosome-lysosome fusion and are mutually exclusive subunits in a SyxSnap29 complex, these data suggest that Vamp7 is directly involved in membrane fusion and Ykt6 acts as a non-conventional, regulatory SNARE in this process. Circadian clocks have evolved as time-measuring molecular devices to help organisms adapt their physiology to daily changes in light and temperature. Transcriptional oscillations account for a large fraction of rhythmic protein abundance. However, cycling of various posttranslational modifications, such as ubiquitylation, also contributes to shape the rhythmic protein landscape.
In this study, we used an in vivo ubiquitin labeling assay to investigate the circadian ubiquitylated proteome of Drosophila melanogaster. These findings stress the importance of ubiquitylation pathways in the Drosophila circadian clock and reveal a key component of this system. Accordingly, activated Notch tumorigenesis was fueled by hampering the immune response or by NOS overexpression to mimic a protumorigenic environment.
Cotranslational protein folding can facilitate rapid formation of functional structures. However, it can also cause premature assembly of protein complexes, if two interacting nascent chains are in close proximity. By analyzing known protein structures, we show that homomeric protein contacts are enriched toward the C termini of polypeptide chains across diverse proteomes.
We hypothesize that this is the result of evolutionary constraints for folding to occur before assembly. Using high-throughput imaging of protein homomers in Escherichia coli and engineered protein constructs with N- and C-terminal oligomerization domains, we show that, indeed, proteins with C-terminal homomeric interface residues consistently assemble more efficiently than those with N-terminal interface residues.
Using in vivo, in vitro and in silico experiments, we identify features that govern successful assembly of homomers, which have implications for protein design and expression optimization. A key challenge in molecular systems biology is understanding how new pathways arise during evolution and how to exploit them for biotechnological applications. New pathways in metabolic networks often evolve by recruiting weak promiscuous activities of pre-existing enzymes. Here we describe recent systems biology advances to map such 'underground' activities and to predict and analyze their contribution to new metabolic functions.
Underground activities are prevalent in cellular metabolism and can form novel pathways that either enable evolutionary adaptation to new environments or provide bypass to genetic lesions. We also illustrate the potential of integrating computational models of underground metabolism and experimental approaches to study the evolution of novel metabolic phenotypes and advance the field of biotechnology. Rapidly spreading antibiotic resistance and the low discovery rate of new antimicrobial compounds demand more effective strategies for early drug discovery.
One bottleneck in the drug discovery pipeline is the identification of the modes of action MoAs of new compounds. We have developed a rapid systematic metabolome profiling strategy to classify the MoAs of bioactive compounds. The method predicted MoA-specific metabolic responses in the nonpathogenic bacterium Mycobacterium smegmatis after treatment with 62 reference compounds with known MoAs and different metabolic and nonmetabolic targets.
We then analyzed a library of new antimycobacterial compounds with unknown MoAs from a drug discovery effort by the pharmaceutical company GlaxoSmithKline GSK. For six of the GSK compounds with potentially new MoAs, the metabolome profiles suggested their ability to interfere with trehalose and lipid metabolism. This was supported by whole-genome sequencing of spontaneous drug-resistant mutants of the pathogen Mycobacterium tuberculosis and in vitro compound-proteome interaction analysis for one of these compounds. Our compendium of drug-metabolome profiles can be used to rapidly query the MoAs of uncharacterized antimicrobial compounds and should be a useful resource for the drug discovery community.
Proteins are necessary for cellular growth. Concurrently, however, protein production has high energetic demands associated with transcription and translation. Here, we propose that activity of molecular chaperones shape protein burden, that is the fitness costs associated with expression of unneeded proteins. To test this hypothesis, we performed a genome-wide genetic interaction screen in baker's yeast. Impairment of transcription, translation, and protein folding rendered cells hypersensitive to protein burden. Specifically, deletion of specific regulators of the Hspassociated chaperone network increased protein burden.
In agreement with expectation, temperature stress, increased mistranslation and a chemical misfolding agent all substantially enhanced protein burden. Finally, unneeded protein perturbed interactions between key components of the HspHsp90 network involved in folding of native proteins. We conclude that specific chaperones contribute to protein burden. Our work indicates that by minimizing the damaging impact of gratuitous protein overproduction, chaperones enable tolerance to massive changes in genomic expression.
Quantifying heterogeneities within cell populations is important for many fields including cancer research and neurobiology; however, techniques to isolate individual cells are limited. Here, we describe a high-throughput, non-disruptive, and cost-effective isolation method that is capable of capturing individually targeted cells using widely available techniques. Using high-resolution microscopy, laser microcapture microscopy, image analysis, and machine learning, our technology enables scalable molecular genetic analysis of single cells, targetable by morphology or location within the sample.
Menthol is a naturally occurring monoterpene alcohol possessing remarkable biological properties including antipruritic, analgesic, antiseptic, anti-inflammatory and cooling effects. Glycosylation is perhaps the most common post-translational modification. Recently there has been growing interest in cataloging the glycan structures, glycoproteins, and specific sites modified and deciphering the biological functions of glycosylation.
Although the results are piling up for N-glycosylation, O-glycosylation is seriously trailing behind. In our review we reiterate the difficulties researchers have to overcome in order to characterize O-glycosylation. We describe how an ingenious cell engineering method delivered exciting results, and what could we gain from "wild-type" samples. Although we refer to the biological role s of O-glycosylation, we do not provide a complete inventory on this topic. At the onset of metamorphosis, Drosophila salivary gland cells undergo a burst of glue granule secretion to attach the forming pupa to a solid surface.
Here, we show that excess granules evading exocytosis are degraded via direct fusion with lysosomes, a secretory granule-specific autophagic process known as crinophagy. Proper glue degradation within lysosomes also requires the Uvrag-containing Vps34 lipid kinase complex and the v-ATPase proton pump, whereas Atg genes involved in macroautophagy are dispensable for crinophagy. Our work establishes the molecular mechanism of developmentally programmed crinophagy in Drosophila and paves the way for analyzing this process in metazoans.
Significance: Ascorbate Asc is an essential compound both in animals and plants, mostly due to its reducing properties, thereby playing a role in scavenging reactive oxygen species ROS and acting as a cofactor in various enzymatic reactions. Recent Advances: Growing number of evidence shows that excessive Asc accumulation may have negative effects on cellular functions both in humans and plants; inter alia it may negatively affect signaling mechanisms, cellular redox status, and contribute to the production of ROS via the Fenton reaction.
Critical Issues: Both plants and humans tightly control cellular Asc levels, possibly via biosynthesis, transport, and degradation, to maintain them in an optimum concentration range, which, among other factors, is essential to minimize the potentially harmful effects of Asc. On the contrary, the Fenton reaction induced by a high-dose Asc treatment in humans enables a potential cancer-selective cell death pathway. Future Directions: The elucidation of Asc induced cancer selective cell death mechanisms may give us a tool to apply Asc in cancer therapy. On the contrary, the regulatory mechanisms controlling cellular Asc levels are also to be considered, for example, when aiming at generating crops with elevated Asc levels.
Redox Signal. Research performed in the last 5 years has been especially productive and led to significant conceptual changes about the mode of action of these photoreceptors. In this review, we focus on the phytochrome B photoreceptor, the major phytochrome species active in light-grown plants. Finally, we discuss how photobiological properties of phytochrome B enable this photoreceptor to function also as a thermosensor.