RESUMEN
COVID-19, caused by the coronavirus SARS-CoV-2, represents a serious worldwide health issue, with continually emerging new variants challenging current therapeutics. One promising alternate therapeutic avenue is represented by nanobodies, small single-chain antibodies derived from camelids with numerous advantageous properties and the potential to neutralize the virus. For identification and characterization of a broad spectrum of anti-SARS-CoV-2 Spike nanobodies, we further optimized a yeast display method, leveraging a previously published mass spectrometry-based method, using B-cell complementary DNA from the same immunized animals as a source of VHH sequences. Yeast display captured many of the sequences identified by the previous approach, as well as many additional sequences that proved to encode a large new repertoire of nanobodies with high affinities and neutralization activities against different SARS-CoV-2 variants. We evaluated DNA shuffling applied to the three complementarity-determining regions of antiviral nanobodies. The results suggested a surprising degree of modularity to complementarity-determining region function. Importantly, the yeast display approach applied to nanobody libraries from immunized animals allows parallel interrogation of a vast number of nanobodies. For example, we employed a modified yeast display to carry out massively parallel epitope binning. The current yeast display approach proved comparable in efficiency and specificity to the mass spectrometry-based approach, while requiring none of the infrastructure and expertise required for that approach, making these highly complementary approaches that together appear to comprehensively explore the paratope space. The larger repertoires produced maximize the likelihood of discovering broadly specific reagents and those that powerfully synergize in mixtures.
Asunto(s)
Anticuerpos Neutralizantes , SARS-CoV-2 , Anticuerpos de Dominio Único , Animales , Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/genética , Regiones Determinantes de Complementariedad , Saccharomyces cerevisiae/genética , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Anticuerpos de Dominio Único/genética , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
In yeast and animals, cyclin B binds and activates the cyclin-dependent kinase ('CDK') CDK1 to drive entry into mitosis. We show that CYCB1, the sole cyclin B in Chlamydomonas, activates the plant-specific CDKB1 rather than the CDK1 ortholog CDKA1, confirming and extending previous results. Time-lapse microscopy shows that CYCB1 is synthesized before each division in the multiple fission cycle, then is rapidly degraded 3-5 minutes before division occurs. CYCB1 degradation is dependent on the anaphase-promoting complex (APC). Like CYCB1, CDKB1 is not synthesized until late G1; however, CDKB1 is not degraded with each division within the multiple fission cycle, but is degraded after all divisions have ceased. The microtubule plus-end-binding protein EB1 labeled with mNeonGreen allowed detection of mitotic events in live cells. The earliest detectable step in mitosis, splitting of polar EB1 signal into two foci, likely associated with future spindle poles, was dependent on CYCB1. CYCB1-GFP localized close to these foci immediately before spindle formation. Spindle breakdown, cleavage furrow formation and accumulation of EB1 in the furrow were dependent on the APC. In interphase, rapidly growing microtubules are marked by 'comets' of EB1; comets are absent in the absence of APC function. Thus CYCB1/CDKB1 and the APC modulate microtubule function and assembly while regulating mitotic progression. Genetic results suggest an independent additional role for the APC in regulating sister chromatid cohesion; this role is likely conserved across eukaryotes.
Asunto(s)
Chlamydomonas , Ciclina B , Anafase/genética , Ciclosoma-Complejo Promotor de la Anafase/genética , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chlamydomonas/genética , Chlamydomonas/metabolismo , Ciclina B/genética , Ciclina B/metabolismo , Quinasas Ciclina-Dependientes/genética , Mitosis/genética , Huso Acromático/metabolismoRESUMEN
Different intensities of high temperatures affect the growth of photosynthetic cells in nature. To elucidate the underlying mechanisms, we cultivated the unicellular green alga Chlamydomonas reinhardtii under highly controlled photobioreactor conditions and revealed systems-wide shared and unique responses to 24-hour moderate (35°C) and acute (40°C) high temperatures and subsequent recovery at 25°C. We identified previously overlooked unique elements in response to moderate high temperature. Heat at 35°C transiently arrested the cell cycle followed by partial synchronization, up-regulated transcripts/proteins involved in gluconeogenesis/glyoxylate-cycle for carbon uptake and promoted growth. But 40°C disrupted cell division and growth. Both high temperatures induced photoprotection, while 40°C distorted thylakoid/pyrenoid ultrastructure, affected the carbon concentrating mechanism, and decreased photosynthetic efficiency. We demonstrated increased transcript/protein correlation during both heat treatments and hypothesize reduced post-transcriptional regulation during heat may help efficiently coordinate thermotolerance mechanisms. During recovery after both heat treatments, especially 40°C, transcripts/proteins related to DNA synthesis increased while those involved in photosynthetic light reactions decreased. We propose down-regulating photosynthetic light reactions during DNA replication benefits cell cycle resumption by reducing ROS production. Our results provide potential targets to increase thermotolerance in algae and crops.
Asunto(s)
Chlamydomonas reinhardtii , Carbono/metabolismo , Chlamydomonas reinhardtii/genética , Calor , Plantas/metabolismo , Temperatura , Tilacoides/metabolismoRESUMEN
DNA replication is fundamental to all living organisms. In yeast and animals, it is triggered by an assembly of pre-replicative complex including ORC, CDC6 and MCMs. Cyclin Dependent Kinase (CDK) regulates both assembly and firing of the pre-replicative complex. We tested temperature-sensitive mutants blocking Chlamydomonas DNA replication. The mutants were partially or completely defective in DNA replication and did not produce mitotic spindles. After a long G1, wild type Chlamydomonas cells enter a division phase when it undergoes multiple rapid synchronous divisions ('multiple fission'). Using tagged transgenic strains, we found that MCM4 and MCM6 were localized to the nucleus throughout the entire multiple fission division cycle, except for transient cytoplasmic localization during each mitosis. Chlamydomonas CDC6 was transiently localized in nucleus in early division cycles. CDC6 protein levels were very low, probably due to proteasomal degradation. CDC6 levels were severely reduced by inactivation of CDKA1 (CDK1 ortholog) but not the plant-specific CDKB1. Proteasome inhibition did not detectably increase CDC6 levels in the cdka1 mutant, suggesting that CDKA1 might upregulate CDC6 at the transcriptional level. All of the DNA replication proteins tested were essentially undetectable until late G1. They accumulated specifically during multiple fission and then were degraded as cells completed their terminal divisions. We speculate that loading of origins with the MCM helicase may not occur until the end of the long G1, unlike in the budding yeast system. We also developed a simple assay for salt-resistant chromatin binding of MCM4, and found that tight MCM4 loading was dependent on ORC1, CDC6 and MCM6, but not on RNR1 or CDKB1. These results provide a microbial framework for approaching replication control in the plant kingdom.
Asunto(s)
Proteínas de Ciclo Celular/genética , Replicación del ADN/genética , Componente 4 del Complejo de Mantenimiento de Minicromosoma/genética , Mitosis/genética , Animales , División Celular/genética , Núcleo Celular/genética , Chlamydomonas reinhardtii/genética , Quinasas Ciclina-Dependientes/genética , Componente 6 del Complejo de Mantenimiento de Minicromosoma/genética , Mutación/genética , Complejo de Reconocimiento del Origen/genética , Fosforilación/genética , Plásmidos/genética , Proteolisis , Ribonucleótido Reductasas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Many organisms possess multiple and often divergent actins whose regulation and roles are not understood in detail. For example, Chlamydomonas reinhardtii has both a conventional actin (IDA5) and a highly divergent one (NAP1); only IDA5 is expressed in normal proliferating cells. We showed previously that the drug latrunculin B (LatB) causes loss of filamentous (F-) IDA5 and strong up-regulation of NAP1, which then provides essential actin function(s) by forming LatB-resistant F-NAP1. RNA-sequencing analyses now show that this up-regulation of NAP1 reflects a broad transcriptional response, much of which depends on three proteins (LAT1, LAT2, and LAT3) identified previously as essential for NAP1 transcription. Many of the LAT-regulated genes contain a putative cis-acting regulatory site, the "LRE motif." The LatB transcriptional program appears to be activated by loss of F-IDA5 and deactivated by formation of F-NAP1, thus forming an F-actin-dependent negative-feedback loop. Multiple genes encoding proteins of the ubiquitin-proteasome system are among those induced by LatB, resulting in rapid degradation of IDA5 (but not NAP1). Our results suggest that IDA5 degradation is functionally important because nonpolymerizable LatB-bound IDA5 interferes with the formation of F-NAP1. The genes for the actin-interacting proteins cofilin and profilin are also induced. Cofilin induction may further the clearance of IDA5 by promoting the scission of F-IDA5, whereas profilin appears to function in protecting monomeric IDA5 from degradation. This multifaceted regulatory system allows rapid and quantitative turnover of F-actin in response to cytoskeletal perturbations and probably also maintains F-actin homeostasis under normal growth conditions.
Asunto(s)
Actinas/biosíntesis , Chlamydomonas reinhardtii/metabolismo , Proteínas de Plantas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Transcripción Genética , Actinas/genética , Chlamydomonas reinhardtii/genética , Proteínas de Plantas/genética , Complejo de la Endopetidasa Proteasomal/genéticaRESUMEN
Biology emerges from interactions between molecules, which are challenging to elucidate with current techniques. An orthogonal approach is to probe for 'response signatures' that identify specific circuit motifs. For example, bistability, hysteresis, or irreversibility are used to detect positive feedback loops. For adapting systems, such signatures are not known. Only two circuit motifs generate adaptation: negative feedback loops (NFLs) and incoherent feed-forward loops (IFFLs). On the basis of computational testing and mathematical proofs, we propose differential signatures: in response to oscillatory stimulation, NFLs but not IFFLs show refractory-period stabilization (robustness to changes in stimulus duration) or period skipping. Applying this approach to yeast, we identified the circuit dominating cell cycle timing. In Caenorhabditis elegans AWA neurons, which are crucial for chemotaxis, we uncovered a Ca2+ NFL leading to adaptation that would be difficult to find by other means. These response signatures allow direct access to the outlines of the wiring diagrams of adapting systems.
Asunto(s)
Adaptación Fisiológica/fisiología , Retroalimentación Fisiológica/fisiología , Modelos Biológicos , Animales , Caenorhabditis elegans , Ciclo Celular/fisiología , Regulación de la Expresión Génica/fisiología , Neuronas/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
B-type cyclins promote mitotic entry and inhibit mitotic exit. In Saccharomyces cerevisiae, four B-type cyclins, Clb1-4, carry out essential mitotic roles, with substantial but incomplete overlap of function among them. Previous work in many organisms has indicated that B-type cyclin-dependent inhibition of mitotic exit imposes a requirement for mitotic destruction of B-type cyclins. For instance, precise genomic removal of the Clb2 destruction box (D box) prevents mitotic proteolysis of Clb2, and blocks mitotic exit. Here, we show that, despite significant functional overlap between Clb2 and Clb3, D-box-dependent Clb3 proteolysis is completely dispensable for mitotic exit. Removal of the Clb3 D box results in abundant Clb3 protein and associated kinase throughout the cell cycle, but mitotic exit occurs with close to normal timing. Clb3 degradation is required for pre-Start G1 control in the succeeding cell cycle. Deleting the CLB3 D box essentially eliminates all time delay before cell cycle Start following division, even in very small newborn cells. CLB3∆db cells show no cell cycle arrest response to mating pheromone, and CLB3∆db completely bypasses the requirement for CLN G1 cyclins, even in the absence of the early expressed B-type cyclins CLB5,6 Thus, regulated mitotic proteolysis of Clb3 is specifically required to make passage of Start in the succeeding cell cycle "memoryless"-dependent on conditions within that cycle, and independent of events such as B-type cyclin accumulation that occurred in the preceding cycle.
Asunto(s)
Ciclina B/metabolismo , Mitosis/genética , Proteolisis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Ciclina B/genética , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Throughout cell-cycle progression, the expression of multiple transcripts oscillate, and whether these are under the centralized control of the CDK-APC/C proteins or can be driven by a de-centralized transcription factor (TF) cascade is a fundamental question for understanding cell-cycle regulation. In budding yeast, we find that the transcription of nearly all genes, as assessed by RNA-seq or fluorescence microscopy in single cells, is dictated by CDK-APC/C. Three exceptional genes are transcribed in a pulsatile pattern in a variety of CDK-APC/C arrests. Pursuing one of these transcripts, the SIC1 inhibitor of B-type cyclins, we use a combination of mathematical modeling and experimentation to provide evidence that, counter-intuitively, Sic1 provides a failsafe mechanism promoting nuclear division when levels of mitotic cyclins are low.
Asunto(s)
Relojes Biológicos , Ciclo Celular , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Ciclinas/metabolismo , Dineínas/genética , Proteínas de Saccharomyces cerevisiae/genética , Análisis de la Célula IndividualRESUMEN
We describe a highly efficient method for exact gene replacement in budding yeast. Induction of rapid and efficient recombination in an entire cell population results in at least 50% of the recombinants undergoing a switch of the endogenous copy to a specific mutated allele, with no remaining markers or remnant of foreign DNA, without selection. To accomplish this, a partial copy of the replacement allele, followed by an HO cut site, is installed adjacent to the wild-type locus, in a GAL-HO MATa-inc background. HO induction results in near-quantitative site cleavage and recombination/gene conversion, resulting in either regeneration of wild-type or switch of the endogenous allele to the mutant, with accompanying deletion of intervening marker sequences, yielding an exact replacement. Eliminating the need for selection (over days) of rare recombinants removes concerns about second-site suppressor mutations and also allows direct phenotypic analysis, even of lethal gene replacements, without the need of a method to make the lethality conditional or to employ regulated promoters of unknown strength compared to the endogenous promoter. To test this method, we tried two known lethal gene replacements, substituting the non-essential CDH1 gene with a dominantly lethal version mutated for its Cdk phosphorylation sites and substituting the essential CDC28 gene with two recessively lethal versions, one containing an early stop codon and another inactivating Cdc28 kinase activity. We also tested a gene replacement of unknown phenotypic consequences: replacing the non-essential CLB3 B-type cyclin with a version lacking its destruction box.