Biology subjects

Gonzalez, B.

Publications and source records attributed to Gonzalez, B..

5 recordsLinked to original sources

bioRxiv2021

Somatic hypomethylation of pericentromeric SST1 repeats and tetraploidization in human colorectal cancer cells

Somatic DNA hypomethylation and aneuploidy are hallmarks of cancer, and there is evidence for a causal relationship between them in knockout mice, but not in human cancer. The non-mobile pericentromeric repetitive elements SST1 are hypomethylated in about 17% of human colorectal cancers (CRC) with some 5-7% exhibiting a more severe age-independent demethylation. Tetraploidy is a common and early event in solid tumors generating subsequent aneuploidy. We compared the relative frequency of chromosomal variations during culture of randomly selected single cell clones of diploid LS174T human CRC cells differing in their levels of SST1 demethylation. Diploid cells underwent frequent genome reduplication events generating tetraploid clones that correlated with SST1 demethylation. In primary CRC, severe SST1 hypomethylation was significantly associated with global genomic hypomethylation and mutations in TP53. This work uncovers the association of the naturally occurring demethylation of the SST1 pericentromeric repeat with the onset of spontaneous tetraploidization in human CRC cells in culture, and with TP53 mutations in primary CRCs. Altogether, our findings provide further support for an oncogenic pathway linking somatic epigenetic and genetic alterations in a subset of human CRC.

cancer biology↗

bioRxiv2020

Spatiotemporal Control of Pathway Sensors and Cross-Pathway Feedback Regulate a Cell Differentiation MAPK Pathway in Yeast

Mitogen-Activated Protein Kinase (MAPK) pathways control cell differentiation and the response to stress. MAPK pathways can share components with other pathways yet induce specific responses through mechanisms that remain unclear. In Saccharomyces cerevisiae, the MAPK pathway that controls filamentous growth (fMAPK) shares components with the MAPK pathway that regulates the response to osmotic stress (HOG). By exploring temporal regulation of MAPK signaling, we show here that the two pathways exhibited different patterns of activity throughout the cell cycle. The different patterns resulted from different expression profiles of genes encoding the mucin sensors (MSB2 for fMAPK and HKR1 for HOG). We also show that positive feedback through the fMAPK pathway stimulated the HOG pathway, presumably to modulate fMAPK pathway activity. By exploring spatial regulation of MAPK signaling, we found that the shared tetraspan protein, Sho1p, which has a dynamic localization pattern, induced the fMAPK pathway at the mother-bud neck. A Sho1p-interacting protein, Hof1p, which also localizes to the mother-bud neck and regulates cytokinesis, also regulated the fMAPK pathway. Therefore, spatial and temporal regulation of pathway sensors, and cross-pathway feedback, regulate a MAPK pathway that controls a cell differentiation response in yeast.

cell biology↗

bioRxiv2020

Pilot production of SARS-CoV-2 related proteins in plants: a proof of concept for rapid repurposing of indoors farms into biomanufacturing facilities

The current CoVid-19 crisis is revealing the strengths and the weaknesses of the worlds capacity to respond to a global health crisis. A critical weakness has resulted from the excessive centralization of the current biomanufacturing capacities, a matter of great concern, if not a source of nationalistic tensions. On the positive side, scientific data and information have been shared at an unprecedented speed fuelled by the preprint phenomena, and this has considerably strengthened our ability to develop new technology-based solutions. In this work we explore how, in a context of rapid exchange of scientific information, plant biofactories can serve as a rapid and easily adaptable solution for local manufacturing of bioreagents, more specifically recombinant antibodies. For this purpose, we tested our ability to produce, in the framework of an academic lab and in a matter of weeks, milligram amounts of six different recombinant monoclonal antibodies against SARS-CoV-2 in Nicotiana benthamiana. For the design of the antibodies we took advantage, among other data sources, of the DNA sequence information made rapidly available by other groups in preprint publications. mAbs were all engineered as single-chain fragments fused to a human gamma Fc and transiently expressed using a viral vector. In parallel, we also produced the recombinant SARS-CoV-2 N protein and its Receptor Binding Domain (RBD) in planta and used them to test the binding specificity of the recombinant mAbs. Finally, for two of the antibodies we assayed a simple scale-up production protocol based on the extraction of apoplastic fluid. Our results indicate that gram amounts of anti-SARS-CoV-2 antibodies could be easily produced in little more than 6 weeks in repurposed greenhouses with little infrastructure requirements using N. benthamiana as production platform. Similar procedures could be easily deployed to produce diagnostic reagents and, eventually, could be adapted for rapid therapeutic responses.

synthetic biology↗

bioRxiv2020

Arabidopsis thaliana interaction with Ensifer meliloti can support plant growth under N-deficiency

Nitrogen (N) is an essential macronutrient for plants. Some plant species obtain this nutrient by interacting with N-fixing bacteria. These beneficial interactions are well described in legumes but have also been observed in non-legume plant species that are unable to form root nodules. We studied the expanding role of beneficial plant-bacteria interactions for N-nutrition in the widely used model plant Arabidopsis thaliana. We found that the bacteria Ensifer meliloti enhanced A. thaliana growth under severe N-deficiency conditions, allowing plants to complete their life cycle. Our results showed that bacteria colonize the rhizosphere associated with the epidermis of the plant root. We also demonstrated that A. thaliana possesses genes that are critical for this beneficial interaction and are required for plant-growth promotion by E. meliloti under N-deficiency. This work shows association between A. thaliana and E. meliloti for plant nutrition under severe N-deficiency, and suggests that plants have conserved-molecular mechanisms to interact with N-fixing bacteria to procure N and escape adverse conditions. Under these circumstances, the supply of N via N-fixation is critical for survival, allowing the plant to complete its life cycle. Our findings provide a new framework and an experimental model system that expand our understanding of plant-rhizobia interactions for plant N-nutrition.

plant biology↗

bioRxiv2019

Functions for Cdc42p BEM Adaptors in Regulating a Differentiation-Type MAP Kinase Pathway

Rho GTPases regulate cell polarity and signal transduction pathways to control morphogenetic responses in different settings. In yeast, the Rho GTPase Cdc42p regulates cell polarity, and through the p21-activated kinase Ste20p, Cdc42p also regulates mitogen-activated protein kinase (MAPK) pathways (mating, filamentous growth or fMAPK, and HOG). Although much is known about how Cdc42p regulates cell polarity and the mating pathway, how Cdc42p regulates the fMAPK pathway is not clear. To address this question, Cdc42p-dependent MAPK pathways were compared in the filamentous ({sum}1278b) strain background. Each MAPK pathway showed a unique activation profile, with the fMAPK pathway exhibiting slow activation kinetics compared to the mating and HOG pathways. A previously characterized version of Cdc42p, Cdc42pE100A, that is specifically defective for fMAPK pathway signaling, was defective for interaction with Bem4p, the pathway-specific adaptor for the fMAPK pathway. Corresponding residues in Bem4p were identified that were required for interaction with Cdc42p and fMAPK pathway signaling. The polarity adaptor Bem1p also regulated the fMAPK pathway. In the fMAPK pathway, Bem1p recruited Ste20p to the plasma membrane, cycled between an open and closed conformation, and interacted with the GEF for Cdc42, Cdc24p. Bem1p also regulated effector pathways in different ways, behaving as a multi-functional adaptor in some pathways and an inert scaffold in others. Genetic suppression tests showed that Bem4p and Bem1p regulate the fMAPK pathway in an ordered sequence. Collectively, the study demonstrates unique and sequential functions for Rho GTPase adaptors in regulating MAPK pathways.\n\nHIGHLIGHTSO_LIComparing Cdc42p-dependent MAPK pathways showed that the fMAPK pathway had slow activation kinetics compared to the mating and HOG pathways.\nC_LIO_LIA collection of cdc42 alleles was tested for MAPK pathway functions.[§] Cdc42pE100A, previously characterized as being specifically defective for fMAPK signaling, showed reduced interaction with the fMAPK pathway adaptor Bem4p.\n[§]Corresponding residues in Bem4p were identified that were required for interaction with Cdc42p and fMAPK signaling.\n\nC_LIO_LIThe polarity adaptor Bem1p regulated the fMAPK pathway.[§] Bem1p regulated the fMAPK pathway by recruiting Ste20p to the plasma membrane, cycling between an open and closed conformation, and interacting with the Cdc42p GEF, Cdc24p.\n\nC_LIO_LIDifferent domains of Bem1p had different roles in regulating effector pathways.[§] Bem1p may function as a multi-functional adaptor in some pathways and an inert scaffold in others.\n\nC_LIO_LIBem4p and Bem1p regulated the fMAPK pathway in an ordered sequence.[§] The data support a model where Bem4p recruits Cdc24p to GDP-Cdc42p, and Bem1p directs GTP-Cdc42p to Ste20p at the plasma membrane.\n[§]The bud-site GTPase Rsr1p regulates Cdc24p in the fMAPK pathway but does not initiate signaling.\n\nC_LI

genetics↗