Chemical genetic analysis of FTY720‐ and Ca 2+ ‐sensitive mutants reveals a functional connection between FTY720 and membrane trafficking

2020 ◽  
Author(s):  
Kanako Hagihara ◽  
Yuki Kanda ◽  
Kouki Ishida ◽  
Ryosuke Satoh ◽  
Teruaki Takasaki ◽  
...  
Keyword(s):  
Genetic Analysis ◽  
Membrane Trafficking ◽  
Functional Connection ◽  
Chemical Genetic

scholarly journals Chemical Genetic Analysis of Apg1 Reveals A Non-kinase Role in the Induction of Autophagy

2003 ◽  
Vol 14 (2) ◽  
pp. 477-490 ◽  
Author(s):  
Hagai Abeliovich ◽  
Chao Zhang ◽  
William A. Dunn ◽  
Kevan M. Shokat ◽  
Daniel J. Klionsky
Keyword(s):  
Signal Transduction ◽  
Membrane Trafficking ◽  
Kinase Activity ◽  
Qualitative Difference ◽  
Membrane Dynamics ◽  
Yeast Saccharomyces Cerevisiae ◽  
Direct Signal ◽  
Chemical Genetic ◽  
Trafficking Pathway ◽  
Induction Process

Macroautophagy is a catabolic membrane trafficking phenomenon that is observed in all eukaryotic cells in response to various stimuli, such as nitrogen starvation and challenge with specific hormones. In the yeast Saccharomyces cerevisiae, the induction of autophagy involves a direct signal transduction mechanism that affects membrane dynamics. In this system, the induction process modifies a constitutive trafficking pathway called the cytoplasm-to-vacuole targeting (Cvt) pathway, which transports the vacuolar hydrolase aminopeptidase I, from the formation of small Cvt vesicles to the formation of autophagosomes. Apg1 is one of the proteins required for the direct signal transduction cascade that modifies membrane dynamics. Although Apg1 is required for both the Cvt pathway and autophagy, we find that Apg1 kinase activity is required only for Cvt trafficking of aminopeptidase I but not for import via autophagy. In addition, the data support a novel role for Apg1 in nucleation of autophagosomes that is distinct from its catalytic kinase activity and imply a qualitative difference in the mechanism of autophagosome and Cvt vesicle formation.

scholarly journals ADP-Ribosylation Factor 6 and a Functional PIX/p95-APP1 Complex Are Required for Rac1B-mediated Neurite Outgrowth

2003 ◽  
Vol 14 (4) ◽  
pp. 1295-1307 ◽  
Author(s):  
Chiara Albertinazzi ◽  
Lorena Za ◽  
Simona Paris ◽  
Ivan de Curtis
Keyword(s):  
Membrane Trafficking ◽  
Functional Connection ◽  
Actin Organization ◽  
Neurite Extension ◽  
Adp Ribosylation ◽  
Carboxy Terminal ◽  
Endocytic Vesicles ◽  
Adp Ribosylation Factor ◽  
Developing Nervous System

The mechanisms coordinating adhesion, actin organization, and membrane traffic during growth cone migration are poorly understood. Neuritogenesis and branching from retinal neurons are regulated by the Rac1B/Rac3 GTPase. We have identified a functional connection between ADP-ribosylation factor (Arf) 6 and p95-APP1 during the regulation of Rac1B-mediated neuritogenesis. P95-APP1 is an ADP-ribosylation factor GTPase-activating protein (ArfGAP) of the GIT family expressed in the developing nervous system. We show that Arf6 has a predominant role in neurite extension compared with Arf1 and Arf5. Cotransfection experiments indicate a specific and cooperative potentiation of neurite extension by Arf6 and the carboxy-terminal portion of p95-APP1. Localization studies in neurons expressing different p95-derived constructs show a codistribution of p95-APP1 with Arf6, but not Arf1. Moreover, p95-APP1–derived proteins with a mutated or deleted ArfGAP domain prevent Rac1B-induced neuritogenesis, leading to PIX-mediated accumulation at large Rab11-positive endocytic vesicles. Our data support a role of p95-APP1 as a specific regulator of Arf6 in the control of membrane trafficking during neuritogenesis.

A Chemical Genetic Analysis of Platelet Activation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4009-4009
Author(s):  
Robert Flaumenhaft ◽  
Lynn VerPlank ◽  
James R. Dilks ◽  
Price S. Blair ◽  
Albert Mairuhu ◽  
...  
Keyword(s):  
Genetic Analysis ◽  
Small Molecules ◽  
Platelet Activation ◽  
Significant Inhibition ◽  
Active Compounds ◽  
Chemical Genetic ◽  
Primary Screening ◽  
Inhibitory Compounds ◽  
Granule Secretion ◽  
Granule Release

Abstract Abstract 4009 Poster Board III-945 Platelets are anucleate cells that are not amenable to traditional forward genetic analysis. In collaboration with the Broad Institute Probe Development Center, we have performed a chemical genetic analysis of platelet activation. Chemical genetics involves exposure of cells to a library of small molecules, identification of compounds that produce a phenotype of interest, and determination of the target of these small molecules. We have used an assay designed both to identify allosteric inhibitors of Protease Activated Receptor-1 (PAR1) and to find inhibitors that selectively target granule release. This assay monitors dense granule secretion mediated by SFLLRN, a PAR1-specific agonist, using a luciferase-based assay system to detect ADP/ATP release. For primary screening, over 300,000 compounds were assayed in duplicate using freshly outdated platelet-rich plasma supplied by several blood banks across the United States. Computational analyses of the primary data demonstrated that approximately 0.2% of compounds showed ≥50% inhibition relative to maximally inhibitory concentrations of the known antiplatelet agent, cilostazol. Secondary screening using 8-point dose response curves were performed on the 629 inhibitory compounds, 742 compounds with inconclusive activity (e.g., ambiguous duplicates in primary screening), and 213 structural analogs of active compounds. These assays identified 367 active compounds with IC50s ≤10 micromolar. Counter screening to exclude luciferase inhibitors demonstrated 137 small molecules that inhibited PAR1-mediated ATP/ADP release without significant inhibition of luciferase. Database mining using PubChem and CAS search engines was performed to assess the selectivity of active compounds. Twenty eight compounds were selected for further testing based on their IC50s in confirmatory assays, lack of activity in unrelated bioassays, and chemical structure. Known platelet inhibitors were excluded. Of the 28 compounds, 16 compounds potently inhibited SFLLRN-induced alpha-granule release from washed platelets, as monitored by P-selectin expression. IC50s for these compounds ranged from <0.3 to 1 micromolar. None of the selected compounds that failed to inhibit alpha-granule release demonstrated significant inhibition of SFLLRN-induced 14C-serotonin release at 10 micromolar. Ongoing studies are directed at selecting best candidates from among the 16 confirmed inhibitory compounds to develop as biological probes that target either PAR1 activation or distal steps in granule secretion. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Chemical genetic analysis of the regulatory role of Cdc2p in the S. pombe septation initiation network

2008 ◽  
Vol 121 (6) ◽  
pp. 843-853 ◽  
Author(s):  
S. Dischinger ◽  
A. Krapp ◽  
L. Xie ◽  
J. R. Paulson ◽  
V. Simanis
Keyword(s):  
Genetic Analysis ◽  
Regulatory Role ◽  
Chemical Genetic ◽  
Septation Initiation Network
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