Annie Quan

Inhibition of dynamin mediated endocytosis by the dynoles: synthesis and functional activity of a family of indoles

Screening identified two bisindolylmaleimides as 100 microM inhibitors of the GTPase activity of dynamin I. Focused library approaches allowed development of indole-based dynamin inhibitors called dynoles. 100-Fold in vitro enhancement of potency was noted with the best inhibitor, 2-cyano-3-(1-(2-(dimethylamino)ethyl)-1H-indol-3-yl)-N-octylacrylamide (dynole 34-2), a 1.3 +/- 0.3 microM dynamin I inhibitor. Dynole 34-2 potently inhibited receptor mediated endocytosis (RME) internalization of Texas red-transferrin. The rank order of potency for a variety of dynole analogues on RME in U2OS cells matched their rank order for dynamin inhibition, suggesting that the mechanism of inhibition is via dynamin. Dynoles are the most active dynamin I inhibitors reported for in vitro or RME evaluations. Dynole 34-2 is 15-fold more active than dynasore against dynamin I and 6-fold more active against dynamin mediated RME (IC(50) approximately 15 microM; RME IC(50) approximately 80 microM). The dynoles represent a new series of tools to better probe endocytosis and dynamin-mediated trafficking events in a variety of cells.

Building a Better Dynasore: The Dyngo Compounds Potently Inhibit Dynamin and Endocytosis†

Dynamin GTPase activity increases when it oligomerizes either into helices in the presence of lipid templates or into rings in the presence of SH3 domain proteins. Dynasore is a dynamin inhibitor of moderate potency (IC50 ˜ 15 μM in vitro). We show that dynasore binds stoichiometrically to detergents used for in vitro drug screening, drastically reducing its potency (IC50 = 479 μM) and research tool utility. We synthesized a focused set of dihydroxyl and trihydroxyl dynasore analogs called the Dyngo™ compounds, five of which had improved potency, reduced detergent binding and reduced cytotoxicity, conferred by changes in the position and/or number of hydroxyl substituents. The Dyngo compound 4a was the most potent compound, exhibiting a 37‐fold improvement in potency over dynasore for liposome‐stimulated helical dynamin activity. In contrast, while dynasore about equally inhibited dynamin assembled in its helical or ring states, 4a and 6a exhibited >36‐fold reduced activity against rings, suggesting that they can discriminate between helical or ring oligomerization states. 4a and 6a inhibited dynamin‐dependent endocytosis of transferrin in multiple cell types (IC50 of 5.7 and 5.8 μM, respectively), at least sixfold more potently than dynasore, but had no effect on dynamin‐independent endocytosis of cholera toxin. 4a also reduced synaptic vesicle endocytosis and activity‐dependent bulk endocytosis in cultured neurons and synaptosomes. Overall, 4a and 6a are improved and versatile helical dynamin and endocytosis inhibitors in terms of potency, non‐specific binding and cytotoxicity. The data further suggest that the ring oligomerization state of dynamin is not required for clathrin‐mediated endocytosis.

Myristyl Trimethyl Ammonium Bromide and Octadecyl Trimethyl Ammonium Bromide Are Surface-Active Small Molecule Dynamin Inhibitors that Block Endocytosis Mediated by Dynamin I or Dynamin II

Dynamin is a GTPase enzyme involved in membrane constriction and fission during endocytosis. Phospholipid binding via its pleckstrin homology domain maximally stimulates dynamin activity. We developed a series of surface-active small-molecule inhibitors, such as myristyl trimethyl ammonium bromide (MiTMAB) and octadecyltrimethyl ammonium bromide (OcTMAB), and we now show MiTMAB targets the dynamin-phospholipid interaction. MiTMAB inhibited dynamin GTPase activity, with a Ki of 940 ± 25 nM. It potently inhibited receptor-mediated endocytosis (RME) of transferrin or epidermal growth factor (EGF) in a range of cells without blocking EGF binding, receptor number, or autophosphorylation. RME inhibition was rapidly reversed after washout. The rank order of potency for a variety of MiTMAB analogs on RME matched the rank order for dynamin inhibition, suggesting dynamin recruitment to the membrane is a primary cellular target. MiTMAB also inhibited synaptic vesicle endocytosis in rat brain nerve terminals (synaptosomes) without inducing depolarization or morphological defects. Therefore, the drug rapidly and reversibly blocks multiple forms of endocytosis with no acute cellular damage. The unique mechanism of action of MiTMAB provides an important tool to better understand dynamin-mediated membrane trafficking events in a variety of cells.

Rapid Purification of Native Dynamin I and Colorimetric GTPase Assay

Dynamin I is a large GTPase enzyme required in membrane constriction and fission during multiple forms of endocytosis. The first method described here is for the rapid purification of native dynamin from peripheral membrane extracts of sheep brain using ammonium sulfate precipitation and affinity purification on recombinant SH3 domains. The method greatly enriches for dynamin I at high purity and allows for large-scale biochemical and functional studies. The second method is a nonradioactive, high-throughput colorimetric GTPase assay for dynamin activity. The approach is based on terminating incubations with EDTA and the use of malachite green for high-sensitivity detection of inorganic phosphate release. The two methods will facilitate high-throughput screens for potential dynamin inhibitors or activators.

PICK1 interacts with PACSIN to regulate AMPA receptor internalization and cerebellar long-term depression

The dynamic trafficking of AMPA receptors (AMPARs) into and out of synapses is crucial for synaptic transmission, plasticity, learning, and memory. The protein interacting with C-kinase 1 (PICK1) directly interacts with GluA2/3 subunits of the AMPARs. Although the role of PICK1 in regulating AMPAR trafficking and multiple forms of synaptic plasticity is known, the exact molecular mechanisms underlying this process remain unclear. Here, we report a unique interaction between PICK1 and all three members of the protein kinase C and casein kinase II substrate in neurons (PACSIN) family and show that they form a complex with AMPARs. Our results reveal that knockdown of the neuronal-specific protein, PACSIN1, leads to a significant reduction in AMPAR internalization following the activation of NMDA receptors in hippocampal neurons. The interaction between PICK1 and PACSIN1 is regulated by PACSIN1 phosphorylation within the variable region and is required for AMPAR endocytosis. Similarly, the binding of PICK1 to the ubiquitously expressed PACSIN2 is also regulated by the homologous phosphorylation sites within the PACSIN2-variable region. Genetic deletion of PACSIN2, which is highly expressed in Purkinje cells, eliminates cerebellar long-term depression. This deficit can be fully rescued by overexpressing wild-type PACSIN2, but not by a PACSIN2 phosphomimetic mutant, which does not bind PICK1 efficiently. Taken together, our data demonstrate that the interaction of PICK1 and PACSIN is required for the activity-dependent internalization of AMPARs and for the expression of long-term depression in the cerebellum.

Phosphorylation of dynamin II at serine-764 is associated with cytokinesis.

Calcineurin is a phosphatase that is activated at the last known stage of mitosis, abscission. Among its many substrates, it dephosphorylates dynamin II during cytokinesis at the midbody of dividing cells. However, dynamin II has several cellular roles including clathrin-mediated endocytosis, centrosome cohesion and cytokinesis. It is not known whether dynamin II phosphorylation plays a role in any of these functions nor have the phosphosites involved in cytokinesis been directly identified. We now report that dynamin II from rat lung is phosphorylated to a low stoichiometry on a single major site, Ser-764, in the proline-rich domain. Phosphorylation on Ser-764 also occurred in asynchronously growing HeLa cells and was greatly increased upon mitotic entry. Tryptic phospho-peptides isolated by TiO(2) chromatography revealed only a single phosphosite in mitotic cells. Mitotic phosphorylation was abolished by roscovitine, suggesting the mitotic kinase is cyclin-dependent kinase 1. Cyclin-dependent kinase 1 phosphorylated full length dynamin II and Glutathione-S-Transferase-tagged-dynamin II-proline-rich domain in vitro, and mutation of Ser-764 to alanine reduced proline-rich domain phosphorylation by 80%, supporting that there is only a single major phosphosite. Ser-764 phosphorylation did not affect clathrin-mediated endocytosis or bulk endocytosis using penetratin-based phospho-deficient or phospho-mimetic peptides or following siRNA depletion/rescue experiments. Phospho-dynamin II was enriched at the mitotic centrosome, but this targeting was unaffected by the phospho-deficient or phospho-mimetic peptides. In contrast, the phospho-mimetic peptide displaced endogenous dynamin II, but not calcineurin, from the midbody and induced cytokinesis failure. Therefore, phosphorylation of dynamin II primarily occurs on a single site that regulates cytokinesis downstream of calcineurin, rather than regulating endocytosis or centrosome function.

Phosphorylation of syndapin I F-BAR domain at two helix-capping motifs regulates membrane tubulation

Syndapin I (PACSIN 1) is a synaptically enriched membrane tubulating protein that plays important roles in activity-dependent bulk endocytosis and neuronal morphogenesis. While syndapin I is an in vitro phosphoprotein, it is not known to be phosphorylated in neurons. Here, we report the identification of two phosphorylation sites, S76 and T181, of syndapin I from nerve terminals. Both residues are located at the N-terminal helix-capping motifs (N-Cap) of different α-helices in the F-BAR domain, important for F-BAR homodimer curvature and dimer-dimer filament assembly, respectively. Phospho-mimetic mutations of these residues regulate lipid-binding and tubulation both in vitro and in cells. Neither phosphosite regulated syndapin I function in activity-dependent bulk endocytosis. Rather, T181 phosphorylation was developmentally regulated and inhibited syndapin I function in neuronal morphogenesis. This suggests a novel mechanism for phosphorylation control of an F-BAR function through the regulation of α-helix interactions and stability within the folded F-BAR domain.

Syndapin – a membrane remodelling and endocytic F‐BAR protein

Syndapin [also called PACSIN (protein kinase C and casein kinase II interacting protein)] is an Fes‐CIP4 homology Bin‐amphiphysin‐Rvs161/167 (F‐BAR) and Src‐homology 3 domain‐containing protein. Three genes give rise to three main isoforms in mammalian cells. They each function in different endocytic and vesicle trafficking pathways and provide critical links between the cytoskeletal network in different cellular processes, such as neuronal morphogenesis and cell migration. The membrane remodelling activity of syndapin via its F‐BAR domain and its interaction partners, such as dynamin and neural Wiskott–Aldrich syndrome protein binding to its Src‐homology 3 domain, are important with respect to its function. Its various partner proteins provide insights into its mechanism of action, as well as its differential roles in these cellular processes. Signalling pathways leading to the regulation of syndapin function by phosphorylation are now contributing to our understanding of the broader functions of this family of proteins.

Repurposing molecular mechanisms of transmitter release: a new job for syndapin at the fusion pore. Focus on “Syndapin 3 modulates fusion pore expansion in mouse neuroendocrine chromaffin cells”

when a hormone- or neurotransmitter-filled vesicle reaches the plasma membrane and the stimulus conditions are right, the two membranes kiss. The kiss results in release of the vesicle contents by exocytosis by one of two modes, determined by a fusion pore. The fusion pore is a protein-lipid complex

Depolarization-dependent syndapin i phosphorylation in nerve terminals

This free journal suppl. entitled: Special Issue: 25th Biennial Meeting of the International Society for Neurochemistry jointly with the 13th Meeting of the Asian-Pacific Society for Neurochemistry in conjunction with the 35th Meeting of the Australasian Neuroscience Society 23–27 August 2015, Cairns, AustraliaThis free journal suppl. entitled: Special Issue: 25th Biennial Meeting of the International Society for Neurochemistry jointly with the 13th Meeting of the Asian-Pacific Society for Neurochemistry in conjunction with the 35th Meeting of the Australasian Neuroscience Society 23–27 August 2015, Cairns, Australia