scholarly journals A zebrafish forward genetic screen identifies an indispensable threonine residue in the kinase domain of PRKD2

Biology Open ◽  
2021 ◽  
Vol 10 (3) ◽  
Author(s):  
Panagiota Giardoglou ◽  
Despina Bournele ◽  
Misun Park ◽  
Stavroula Kanoni ◽  
George V. Dedoussis ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Calcineurin Inhibitor ◽  
Kinase Domain ◽  
Genetic Screen ◽  
Outflow Tract ◽  
Forward Genetic Screen ◽  
Evolutionarily Conserved ◽  
Intracardiac Flow ◽  
Conserved Residue ◽  
Higher Sensitivity

ABSTRACT Protein kinase D2 belongs to a family of evolutionarily conserved enzymes regulating several biological processes. In a forward genetic screen for zebrafish cardiovascular mutants, we identified a mutation in the prkd2 gene. Homozygous mutant embryos develop as wild type up to 36 h post-fertilization and initiate blood flow, but fail to maintain it, resulting in a complete outflow tract stenosis. We identified a mutation in the prkd2 gene that results in a T757A substitution at a conserved residue in the kinase domain activation loop (T714A in human PRKD2) that disrupts catalytic activity and drives this phenotype. Homozygous mutants survive without circulation for several days, allowing us to study the extreme phenotype of no intracardiac flow, in the background of a functional heart. We show dysregulation of atrioventricular and outflow tract markers in the mutants and higher sensitivity to the Calcineurin inhibitor, Cyclosporin A. Finally we identify TBX5 as a potential regulator of PRKD2. Our results implicate PRKD2 catalytic activity in outflow tract development in zebrafish. This article has an associated First Person interview with the first author of the paper.

scholarly journals First person – Panagiota Giardoglou and Despina Bournele

Biology Open ◽  
2021 ◽  
Vol 10 (3) ◽  
Keyword(s):  
Developmental Biology ◽  
Cardiovascular Diseases ◽  
Kinase Domain ◽  
Genetic Screen ◽  
Early Career ◽  
First Person ◽  
Forward Genetic Screen ◽  
Threonine Residue ◽  
Early Career Researchers ◽  
Selection Of

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Panagiota Giardoglou and Despina Bournele are co-first authors on ‘A zebrafish forward genetic screen identifies an indispensable threonine residue in the kinase domain of PRKD2’, published in BiO. Panagiota is a PhD student in the lab of Dr Dimitris Beis at the Biomedical Research Foundation Academy of Athens, Greece, investigating modelling human cardiovascular diseases and underlying the mechanisms involved in their pathophysiology. Despina is a toxicologist in the lab of Dr Kyriaki Machera at the Benaki Phytopathological Institute, Kifissia, Athens, Greece, investigating molecular and developmental biology, and zebrafish toxicology.

scholarly journals Massively parallel in vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nathan J. VanDusen ◽  
Julianna Y. Lee ◽  
Weiliang Gu ◽  
Catalina E. Butler ◽  
Isha Sethi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Genetic Screen ◽  
Forward Genetics ◽  
Epigenetic Mark ◽  
Biological Processes ◽  
Dynamic Changes ◽  
Forward Genetic Screen ◽  
High Resource ◽  
Organ Systems

AbstractThe forward genetic screen is a powerful, unbiased method to gain insights into biological processes, yet this approach has infrequently been used in vivo in mammals because of high resource demands. Here, we use in vivo somatic Cas9 mutagenesis to perform an in vivo forward genetic screen in mice to identify regulators of cardiomyocyte (CM) maturation, the coordinated changes in phenotype and gene expression that occur in neonatal CMs. We discover and validate a number of transcriptional regulators of this process. Among these are RNF20 and RNF40, which form a complex that monoubiquitinates H2B on lysine 120. Mechanistic studies indicate that this epigenetic mark controls dynamic changes in gene expression required for CM maturation. These insights into CM maturation will inform efforts in cardiac regenerative medicine. More broadly, our approach will enable unbiased forward genetics across mammalian organ systems.

scholarly journals p50Cdc37 Can Buffer the Temperature-Sensitive Properties of a Mutant of Hck

2000 ◽  
Vol 20 (18) ◽  
pp. 6984-6995 ◽  
Author(s):  
Glen Scholz ◽  
Steven D. Hartson ◽  
Kellie Cartledge ◽  
Nathan Hall ◽  
Jieya Shao ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Active Form ◽  
Tumor Development ◽  
Kinase Domain ◽  
Cellular Level ◽  
Src Family Kinases ◽  
Temperature Sensitive ◽  
Rate Limiting Step ◽  
Truncation Mutant ◽  
Wild Type Counterpart

ABSTRACT Genetic studies have previously revealed that Cdc37p is required for the catalytic competence of v-Src in yeast. We have reasoned that temperature-sensitive mutants of Src family kinases might be more sensitive to the cellular level of p50Cdc37, the mammalian homolog of Cdc37p, than their wild-type counterpart, thus potentially providing a unique opportunity to elucidate the involvement of p50Cdc37 in the folding and stabilization of Src family kinases. A temperature-sensitive mutant of a constitutively active form of Hck (i.e., tsHck499F) was created by mutating two amino acids within the kinase domain of Hck499F. Significantly, overexpression of p50Cdc37 rescues the catalytic activity of tsHck499F at 33°C, while partially buffering it against inactivation at higher temperatures (e.g., 37 and 39°C). Hsp90 function is required for tsHck499F activity and its stabilization by p50Cdc37, but overexpression of Hsp90 is not sufficient to stabilize tsHck499F. Overexpression of p50Cdc37 promotes the association of tsHck499F with Hsp90, suggesting that the cellular level of p50Cdc37might be the rate-limiting step in the association oftsHck499F with Hsp90. A truncation mutant of p50Cdc37 that cannot bind Hsp90 still has a limited capacity to rescue the catalytic activity of tsHck499F and promote its association with Hsp90. This is a particularly important observation, since it argues that rather than solely acting as a passive adapter protein to tether tsHck499F to Hsp90, p50Cdc37 may also act allosterically to enhance the association of tsHck499F with Hsp90. The findings presented here might also have implications for our understanding of the evolution of protein kinases and tumor development.

Abstract LB247: A forward genetic screen to identify drivers of neuroendocrine prostate cancer

2021 ◽  
Author(s):  
Francisca Nunes de Almeida ◽  
Alessandro Vasciaveo ◽  
Min Zou ◽  
Matteo Di Bernardo ◽  
Andrea Califano ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Genetic Screen ◽  
Forward Genetic Screen ◽  
Neuroendocrine Prostate Cancer

scholarly journals Forward genetic screen of human transposase genomic rearrangements

BMC Genomics ◽  
2016 ◽  
Vol 17 (1) ◽  
Author(s):  
Anton G. Henssen ◽  
Eileen Jiang ◽  
Jiali Zhuang ◽  
Luca Pinello ◽  
Nicholas D. Socci ◽  
...  
Keyword(s):  
Genetic Screen ◽  
Genomic Rearrangements ◽  
Forward Genetic Screen

scholarly journals Regulation of Yeast-to-Hyphae Transition inYarrowia lipolytica

mSphere ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
Kyle R. Pomraning ◽  
Erin L. Bredeweg ◽  
Eduard J. Kerkhoven ◽  
Kerrie Barry ◽  
Sajeet Haridas ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Stress Response ◽  
Hyphal Growth ◽  
Genetic Screen ◽  
Morphological Transition ◽  
Histidine Kinases ◽  
Content Type ◽  
Forward Genetic Screen ◽  
Response To Stress

ABSTRACTThe yeastYarrowia lipolyticaundergoes a morphological transition from yeast-to-hyphal growth in response to environmental conditions. A forward genetic screen was used to identify mutants that reliably remain in the yeast phase, which were then assessed by whole-genome sequencing. All thesmoothmutants identified, so named because of their colony morphology, exhibit independent loss of DNA at a repetitive locus made up of interspersed ribosomal DNA and short 10- to 40-mer telomere-like repeats. The loss of repetitive DNA is associated with downregulation of genes with stress response elements (5′-CCCCT-3′) and upregulation of genes with cell cycle box (5′-ACGCG-3′) motifs in their promoter region. The stress response element is bound by the transcription factor Msn2p inSaccharomyces cerevisiae. We confirmed that theY. lipolyticamsn2(Ylmsn2) ortholog is required for hyphal growth and found that overexpression of Ylmsn2enables hyphal growth insmoothstrains. The cell cycle box is bound by the Mbp1p/Swi6p complex inS. cerevisiaeto regulate G1-to-S phase progression. We found that overexpression of either the Ylmbp1or Ylswi6homologs decreased hyphal growth and that deletion of either Ylmbp1or Ylswi6promotes hyphal growth insmoothstrains. A second forward genetic screen for reversion to hyphal growth was performed with thesmooth-33mutant to identify additional genetic factors regulating hyphal growth inY. lipolytica. Thirteen of the mutants sequenced from this screen had coding mutations in five kinases, including the histidine kinases Ylchk1and Ylnik1and kinases of the high-osmolarity glycerol response (HOG) mitogen-activated protein (MAP) kinase cascade Ylssk2, Ylpbs2, and Ylhog1. Together, these results demonstrate thatY. lipolyticatransitions to hyphal growth in response to stress through multiple signaling pathways.IMPORTANCEMany yeasts undergo a morphological transition from yeast-to-hyphal growth in response to environmental conditions. We used forward and reverse genetic techniques to identify genes regulating this transition inYarrowia lipolytica. We confirmed that the transcription factor Ylmsn2is required for the transition to hyphal growth and found that signaling by the histidine kinases Ylchk1and Ylnik1as well as the MAP kinases of the HOG pathway (Ylssk2, Ylpbs2, and Ylhog1) regulates the transition to hyphal growth. These results suggest thatY. lipolyticatransitions to hyphal growth in response to stress through multiple kinase pathways. Intriguingly, we found that a repetitive portion of the genome containing telomere-like and rDNA repeats may be involved in the transition to hyphal growth, suggesting a link between this region and the general stress response.

scholarly journals UNC-2 CaV2 channel localization at presynaptic active zones depends on UNC-10/RIM and SYD-2/Liprin-α in Caenorhabditis elegans

2021 ◽  
Author(s):  
Kelly H. Oh ◽  
Mia Krout ◽  
Janet E. Richmond ◽  
Hongkyun Kim
Keyword(s):  
Active Zone ◽  
Calcium Influx ◽  
Genetic Screen ◽  
Scaffolding Protein ◽  
Tight Coupling ◽  
Forward Genetic Screen ◽  
Precise Control ◽  
C Elegans ◽  
Vesicle Exocytosis ◽  
Active Zones

AbstractPresynaptic active zone proteins couple calcium influx with synaptic vesicle exocytosis. However, the control of presynaptic calcium channel clustering by active zone proteins is not completely understood. In a C. elegans forward genetic screen, we find that UNC-10/RIM (Rab3-interacting molecule) and SYD-2/Liprin-α regulate presynaptic clustering of UNC-2, the CaV2 channel ortholog. We further quantitatively analyzed live animals using endogenously GFP-tagged UNC-2 and active zone components. Consistent with the interaction between RIM and CaV2 in mammals, the intensity and number of UNC-2 channel clusters at presynaptic terminals were greatly reduced in unc-10 mutant animals. To understand how SYD-2 regulates presynaptic UNC-2 channel clustering, we analyzed presynaptic localization of endogenous SYD-2, UNC-10, RIMB-1/RIM-BP (RIM binding protein), and ELKS-1. Our analysis revealed that while SYD-2 is the most critical for active zone assembly, loss of SYD-2 function does not completely abolish presynaptic localization of UNC-10, RIMB-1, and ELKS-1, suggesting an existence of SYD-2-independent active zone assembly. UNC-2 localization analysis in double and triple mutants of active zone components show that SYD-2 promotes UNC-2 clustering by partially controlling UNC-10 localization, and ELKS-1 and RIMB-1 also contribute to UNC-2 channel clustering. In addition, we find that core active zone proteins are unequal in their abundance. While the abundance of UNC-10 at the active zone is comparable to UNC-2, SYD-2 and ELKS-1 are twice more and RIMB-1 four times more abundant than UNC-2. Together our data show that UNC-10, SYD-2, RIMB-1, and ELKS-1 control presynaptic UNC-2 channel clustering in redundant yet distinct manners.Significance StatementPrecise control of neurotransmission is dependent on the tight coupling of the calcium influx through voltage-gated calcium channels (VGCCs) to the exocytosis machinery at the presynaptic active zones. However, how these VGCCs are tethered to the active zone is incompletely understood. To understand the mechanism of presynaptic VGCC localization, we performed a C. elegans forward genetic screen and quantitatively analyzed endogenous active zones and presynaptic VGCCs. In addition to RIM (Rab3-interacting molecule), our study finds that SYD-2/Liprin-α is critical for presynaptic localization of VGCCs. Yet, the loss of SYD-2, the master active zone scaffolding protein, does not completely abolish the presynaptic localization of the VGCC, showing that the active zone is a resilient structure assembled by redundant mechanisms.

scholarly journals CRISPR-Cas immunity repressed by a biofilm-activating pathway inPseudomonas aeruginosa

2019 ◽  
Author(s):  
Adair L. Borges ◽  
Bardo Castro ◽  
Sutharsan Govindarajan ◽  
Tina Solvik ◽  
Veronica Escalante ◽  
...  
Keyword(s):  
Genetic Screen ◽  
Type I ◽  
Two Component System ◽  
Component System ◽  
Forward Genetic Screen ◽  
Immune Systems ◽  
Adaptive Immune ◽  
Biofilm Production ◽  
Two Component ◽  
Adaptive Immune Systems

CRISPR-Cas systems are adaptive immune systems that protect bacteria from bacteriophage (phage) infection. To provide immunity, RNA-guided protein surveillance complexes recognize foreign nucleic acids, triggering their destruction by Cas nucleases. While the essential requirements for immune activity are well understood, the physiological cues that regulate CRISPR-Cas expression are not. Here, a forward genetic screen identifies a two-component system (KinB/AlgB), previously characterized in regulatingPseudomonas aeruginosavirulence and biofilm establishment, as a regulator of the biogenesis and activity of the Type I-F CRISPR-Cas system. Downstream of the KinB/AlgB system, activators of biofilm production AlgU (a σEorthologue) and AlgR, act as repressors of CRISPR-Cas activity during planktonic and surface-associated growth. AmrZ, another biofilm activator, functions as a surface-specific repressor of CRISPR-Cas immunity.Pseudomonasphages and plasmids have taken advantage of this regulatory scheme, and carry hijacked homologs of AmrZ, which are functional CRISPR-Cas repressors. This suggests that while CRISPR-Cas regulation may be important to limit self-toxicity, endogenous repressive pathways represent a vulnerability for parasite manipulation.

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