ABSTRACT
The functions of the actin cytoskeleton in post-Golgi trafficking are still poorly understood. Here, we report the role of LIM Kinase 1 (LIMK1) and its substrate cofilin in the trafficking of apical and basolateral proteins in Madin-Darby canine kidney cells. Our data indicate that LIMK1 and cofilin organize a specialized population of actin filaments at the Golgi complex that is selectively required for the emergence of an apical cargo route to the plasma membrane (PM). Quantitative pulse-chase live imaging experiments showed that overexpression of kinase-dead LIMK1 (LIMK1-KD), or of LIMK1 small interfering RNA, or of an activated cofilin mutant (cofilin S3A), selectively slowed down the exit from the trans-Golgi network (TGN) of the apical PM marker p75-green fluorescent protein (GFP) but did not interfere with the apical PM marker glycosyl phosphatidylinositol-YFP or the basolateral PM marker neural cell adhesion molecule-GFP. High-resolution live imaging experiments of carrier formation and release by the TGN and analysis of peri-Golgi actin dynamics using photoactivatable GFP suggest a scenario in which TGN-localized LIMK1-cofilin regulate a population of actin filaments required for dynamin-syndapin-cortactin-dependent generation and/or fission of precursors to p75 transporters.
Subject(s)
Actin Depolymerizing Factors/metabolism , Dynamins/metabolism , Lim Kinases/metabolism , trans-Golgi Network/metabolism , Actin Depolymerizing Factors/genetics , Actins/metabolism , Animals , Biomarkers/metabolism , Cell Line , Cell Polarity , Cytoskeleton/metabolism , Dogs , Dynamins/genetics , Golgi Apparatus/metabolism , Isoenzymes/genetics , Isoenzymes/metabolism , Lim Kinases/genetics , Models, Biological , Neural Cell Adhesion Molecules/genetics , Neural Cell Adhesion Molecules/metabolism , Protein Transport/physiology , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptor, Nerve Growth Factor/genetics , Receptor, Nerve Growth Factor/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
Inspired by the usefulness of small molecules to study membrane traffic, we used high-throughput synthesis and phenotypic screening to discover secramine, a molecule that inhibits membrane traffic out of the Golgi apparatus by an unknown mechanism. We report here that secramine inhibits activation of the Rho GTPase Cdc42, a protein involved in membrane traffic, by a mechanism dependent upon the guanine dissociation inhibitor RhoGDI. RhoGDI binds Cdc42 and antagonizes its membrane association, nucleotide exchange and effector binding. In vitro, secramine inhibits Cdc42 binding to membranes, GTP and effectors in a RhoGDI-dependent manner. In cells, secramine mimics the effects of dominant-negative Cdc42 expression on protein export from the Golgi and on Golgi polarization in migrating cells. RhoGDI-dependent Cdc42 inhibition by secramine illustrates a new way to inhibit Rho GTPases with small molecules and provides a new means to study Cdc42, RhoGDI and the cellular processes they mediate.
Subject(s)
Actins/metabolism , Benzazepines/pharmacology , Golgi Apparatus/drug effects , Guanine Nucleotide Dissociation Inhibitors/pharmacology , Oximes/pharmacology , cdc42 GTP-Binding Protein/antagonists & inhibitors , Animals , Benzazepines/chemical synthesis , Cattle , Cell Cycle Proteins/metabolism , Cell Membrane/metabolism , Dose-Response Relationship, Drug , GTPase-Activating Proteins/metabolism , Golgi Apparatus/metabolism , Guanine Nucleotide Dissociation Inhibitors/chemical synthesis , Oximes/chemical synthesis , Time Factors , cdc42 GTP-Binding Protein/metabolism , rho-Specific Guanine Nucleotide Dissociation InhibitorsABSTRACT
A distinct subpopulation of rat dorsal root sensory (DRG) neurons, termed P-neurons, switch their trophic requirements for survival during development from nerve growth factor (NGF) at embryonic stages to basic fibroblast growth factor (bFGF) just after birth. We investigated in cultured P-neurons the intracellular signaling pathways mediating survival before and after this switch. The NGF-induced survival was completely blocked by either wortmannin (100 nM) or PD98059 (25-50 nM), which selectively inhibit the phosphatidylinositol 3-kinase-AKT (PI3 kinase-AKT) and mitogen-activated kinase kinase extracellular regulated kinase (MEK-ERKs) pathways, respectively. NGF activated AKT and ERKs in single embryonic P-neurons, as assayed by immunofluorescence of phosphorylated proteins. In concordance with the survival assays, wortmannin and PD98059 blocked AKT and ERKs activation, respectively. Following the trophic switch, bFGF used the same signaling pathways to promote survival of post-natal P-neurons, as either wortmannin or PD98059 blocked its effect. Also, bFGF activated AKT and ERKs in single P-neurons, and this activation was blocked by the same inhibitors. These results strongly suggest that both pathways concurrently mediate the action of NGF and bFGF during embryonic and post-natal periods, respectively. Thus, we report the novel result that the switch in trophic requirements occurs with conservation of the signaling pathways mediating survival.
