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Regulation of dynamin-2 assembly-disassembly and function through the SH3A domain of intersectin-1s - PubMed

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Regulation of dynamin-2 assembly-disassembly and function through the SH3A domain of intersectin-1s

Ivana Knezevic et al. J Cell Mol Med. 2011 Nov.

Abstract

Intersectin-1s (ITSN-1s), a five Src homology 3 (SH3) domain-containing protein, is critically required for caveolae and clathrin-mediated endocytosis (CME), due to its interactions with dynamin (dyn). Of the five SH3A-E domains, SH3A is unique because of its high affinity for dyn and potent inhibition of CME. However, the molecular mechanism by which SH3A integrates in the overall function of ITSN-1s to regulate the endocytic process is not understood. Using biochemical and functional approaches as well as high-resolution electron microscopy, we show that SH3A exogenously expressed in human lung endothelial cells caused abnormal endocytic structures, distorted caveolae clusters, frequent staining-dense rings around the caveolar necks and 60% inhibition of caveolae internalization. In vitro studies further revealed that SH3A, similar to full-length ITSN-1s stimulates dyn2 oligomerization and guanosine triphosphatase (GTP)ase activity, effects not detected when other SH3 domains of ITSN-1s were used as controls. Strikingly, in the presence of SH3A, dyn2-dyn2 interactions are stabilized and despite continuous GTP hydrolysis, dyn2 oligomers cannot disassemble. SH3A may hold up caveolae release from the plasma membrane and formation of free-transport vesicles, by prolonging the lifetime of assembled dyn2. Altogether, our results indicate that ITSN-1s, via its SH3A has the unique ability to regulate dyn2 assembly-disassembly and function during endocytosis.

© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

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Figures

Fig 1
Fig 1

Expression of the SH3A of ITSN-1 in cultured ECs impairs caveolae internalization. (A) In control cells subjected to biotinylation of cell surface proteins and internalization assay (37°C, 30 min.), neutrAvidin-Texas Red staining indicates a strong punctate pattern throughout the cytosol (a1, a1.1), with accumulation in the perinuclear area. ECs transiently transfected with myc-SH3A, selected based on their resistance to blasticidin, and then subjected to the internalization assay using cleavable biotin show limited neutrAvidin-Texas Red staining and some accumulation in the perinuclear area (a2). Anti-myc mAb followed by antimouse IgG Alexa Fluor488-conjugated was used to visualize transfected cells (a3). Use of non-cleavable biotin demonstrates sequestration of biotinylated proteins at the cell surface (a4, a4.1 arrows) and limited internalization. Within cells, biotin was often detected in large puncta under the PM (a4.2, arrows). Bars: 10 μm a1–a4; 5 μm – a1.1, a4.1; 3 μm – a4.2. (B) The number of biotin molecules present in ECs lysates prepared from controls, mock myc-SH3A and myc-SH3E transfected ECs was determined by ELISA in three to four different experiments. Ordinate: number of biotin molecules normalized per mg total protein (TP). Bars ± S.D.

Fig 2
Fig 2

Expression of the SH3A of ITSN-1 in cultured ECs causes abnormal caveolae morphology. (A) The electron micrographs show a segment of a control ECs with omega-shaped caveolae open to the extracellular environment through short necks (a1 arrows, a2) or directly (inset a1.1), and caveolae as apparently detached spherical structures in the cytosol (a1, arrowheads). Clusters of interconnected caveolae are shown in a3, a4. Two clathrin-coated pits open on opposite sides of the PM are shown in a5. Bars: 50 nm. (B) Electron micrographs showing highly magnified caveolar profiles with unusual morphology. Caveolae (b1, b4-b6) and clathrin-coated pits and vesicles (b1, arrowhead, b2) with elongated necks were frequently observed. The micrographs in panels (b7–b11) show staining-dense collars encircling the caveolae necks. Myc-SH3A expression causes unusual caveolae clustering (b1, arrow, b12–b14). Bars: 100 nm (b1); 70 nm (b2–b6); 50 nm (b7, b8, b12–14); 25 nm (b9–b11).

Fig 3
Fig 3

SH3A stimulates dyn2 oligomerization. (A) SH3 pull-down assays were performed with mouse lung lysates (250 μg total protein) and GST-fusion proteins (∼30 μg) encoding the SH3A, E and D, the SH3A-E complement as well as full-length ITSN-1s, coupled to Glutathione Sepharose beads. The proteins bound to the beads were eluted with SDS-PAGE sample buffer, subjected to electrophoresis and electrotransfer to NC membranes. The membranes were probed with anti dyn pAb. (B) Coomassie staining of a 5–20% SDS-PAGE shows the purity of the GST-SH3 fusion proteins obtained by affinity chromatography on Glutathione Sepharose 4B columns from 1 mM IPTG-induced E. coli lysates. Mr: molecular weight markers. (C) GST-SH3A overlay using nitrocellulose membranes containing the ECs proteins (100 μg), resolved by SDS-PAGE were incubated with the 5 μg GST-SH3A (lane SH3A +); nitrocellulose membranes containing ECs proteins from the same experiment were reacted with anti-dyn Ab (lane Dyn2). A direct interaction between dyn2 and ITSN-1s via the SH3A domain is detected. (D) EM– staining showing that purified dyn2 alone (d1), forms high number of rod-like structures and few characteristic rings (d1, white arrow), under physiological conditions (150 mM NaCl). Representative images show increase in the number of dyn2 rings (d2 and boxed area), and a significant increase in the number of dyn2 spirals (d2, arrows) when GST-SH3A is added. Galleries in the inset show high magnification of dyn2 spirals (upper gallery) and dyn2 rings (lower gallery). Bars: 100 nm (d1, d2); 50 nm (gallery insets in d2). (E) Highly magnified, negatively stained dyn2 structures assembled in the absence (e1, e3, e5) and in the presence (e2, e4, e6) of the GST-SH3A. All images are shown at the same magnification. (F) Affinity purified GST-dyn2 (0.1 mg/ml) was assembled into multimers, in the absence or in the presence of GST-SH3 fusion proteins using a low-ionic strength buffer. Oligomeric structures were separated from soluble dyn2 by centrifugation; the pellet (P) and supernatant (S) were analysed by SDS-PAGE followed by Coomassie Blue staining and densitometry. Under control conditions, dyn2 was found in both P and S fractions, slightly more in the S fraction. When GST-SH3A or GST-tagged full-length ITSN-1s are present, the equilibrium is shifted towards the oligomeric state (P fraction). The histogram represents the average amounts of dyn2 in the P and S factions in four different experiments. Densitometric analysis was performed with ImageJ 1.42l, Image Processing Software. (G) 2 μM GST-dyn2 was incubated for 10 min. at RT with increasing amounts of GST-SH3A at physiological salt (150 mM NaCl) concentrations. Samples were subjected to ultracentrifugation and the pellet (P) and supernatant (S) fractions were analysed by SDS-PAGE and Coomassie Blue staining.

Fig 4
Fig 4

GST-SH3A stimulates the GTPase activity of dyn2 and stabilizes dyn2 oligomeric structures. (A) The basal GTPase activity of dyn2 measured in the presence of different GST-SH3 domains of ITSN-1s as well as full length GST-tagged ITSN-1s. (B) Oligomeric dyn2 structures (b1, P fractions) were subjected to GTPase assay in the absence or presence of the GST-SH3A of ITSN-1. The GTPase activity of assembled dyn2 in the presence of GST-SH3A is higher by comparison to dyn2 alone. The GTPase activity of dyn2 decreases as GTP is hydrolysed and the dyn2 rings disassemble. In the presence of GST-SH3A, despite GTP hydrolysis, the GTPase activity of dyn2 remains high and apparently dyn2 conformation does not change. As shown, after 30 min. of GTP hydrolysis, the amount of pelletable dyn2 is significantly high (b2). (C) EM– staining of stabilized dyn2 oligomeric structures, 30 min. after GTP hydrolysis. Dyn2 oligomeric structures – open rings (c1), stacks of rings (c2–c5), are stabilized, apparently unable to change their conformation. Bar: 50 nm.

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