Biology subjects

Garrido, D.

Publications and source records attributed to Garrido, D..

4 recordsLinked to original sources

bioRxiv2020

Cyclin B3 activates the Anaphase-Promoting Complex/Cyclosome in meiosis and mitosis

In mitosis and meiosis, chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit ubiquitin ligase that targets proteins for degradation, leading to the separation of chromatids. APC/C activation requires phosphorylation of its APC3 and APC1 subunits, which allows the APC/C to bind its Cdc20 co-activator. The identity of the kinase(s) responsible for APC/C activation in vivo is unclear. Cyclin B3 is required for meiotic anaphase in flies, worms and vertebrates, but whether it activates the APC/C is unclear. We found that Drosophila Cyclin B3 (CycB3) collaborates with PP2A-B55/Tws in embryonic development, indicating that CycB3 also promotes anaphase in mitosis. Moreover, CycB3 promotes APC/C activity and anaphase in cells in culture. We show that CycB3 physically associates with the APC/C, is required for phosphorylation of APC3, and promotes APC/C association with its co-activators. We propose that CycB3-Cdk1 directly phosphorylates the APC/C to activate it in both meiosis and mitosis.

cell biology↗

bioRxiv2019

Functional Annotation of Human Long Non-Coding RNAs via Molecular Phenotyping

Long non-coding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes and yet, their functions remain largely unknown. We systematically knockdown 285 lncRNAs expression in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNA exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest to-date lncRNA knockdown dataset with molecular phenotyping (over 1,000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.

genomics↗

bioRxiv2019

Differential Metabolic Sensitivity of mTORC1- and mTORC2-Dependent Overgrowth

The glycolytic/lipogenic axis promotes the synthesis of energetic molecules and building blocks necessary to support cell growth, although the absolute requirement of this metabolic axis must be deeply investigated. Here, we used Drosophila genetics and focus on the mTOR signaling network that controls cell growth and homeostasis. mTOR is present in two distinct complexes, mTORC1 and mTORC2. The former directly responds to amino acids and energetic levels, whereas the latter is required to sustain the signaling response downstream of insulin-like-peptide (Ilp) stimulation. Either signaling branch can be independently modulated in most Drosophila tissues. We confirm this independency in the fat tissue. We show that ubiquitous over-activation of mTORC1 or Ilp signaling affects carbohydrate and lipid metabolism, supporting the use of Drosophila as a powerful model to study the link between growth and metabolism. We show that cell-autonomous restriction of glycolysis or lipogenesis in fat cells impedes overgrowth dependent on Ilp-but not mTORC1-signaling. Additionally, ubiquitous deficiency of lipogenesis (FASN mutants) results in a drop in mTORC1 but not Ilp signaling, whereas, at the cell-autonomous level, lipogenesis deficiency affects none of these signals in fat cells. These findings thus, reveal differential metabolic sensitivity of mTORC1- and Ilp-dependent overgrowth. Furthermore, they suggest that local metabolic defects may elicit compensatory pathways between neighboring cells, whereas enzyme knockdown in the whole organism results in animal death. Importantly, our study weakens the use of single inhibitors to fight mTOR-related diseases and strengthens the use of drug combination and selective tissue-targeting.

cell biology↗

bioRxiv2017

Coupling of Polo kinase activation to nuclear localization by a bifunctional NLS is required during mitotic entry.

The Polo kinase is a master regulator of mitosis and cytokinesis conserved from yeasts to humans. Polo is composed of an N-term kinase domain (KD) and a C-term Polo-Box Domain (PBD), which regulates its subcellular localizations. The PBD and KD can interact and inhibit each other, and this reciprocal inhibition is relieved when Polo is phosphorylated at its activation loop. How Polo activation and localization are coupled during mitotic entry is unknown. Here we report that PBD binding to the KD masks a nuclear localization signal (NLS). Activating phosphorylation of the KD leads to exposure of the NLS and entry of Polo into the nucleus before nuclear envelope breakdown. Failures of this mechanism result in misregulation of the Cdk1-activating Cdc25 phosphatase and lead to mitotic and developmental defects in Drosophila. These results uncover novel spatiotemporal mechanisms linking master regulatory enzymes during mitotic entry.

cell biology↗