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First published online 20 June 2006
doi: 10.1242/jcs.03040

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Systematic analysis of myotubularins: heteromeric interactions, subcellular localisation and endosomerelated functions

Physiological Laboratory, University of Liverpool, Crown Street, Liverpool, L69 3BX, UK

View larger version (44K): [in a new window]   Fig. 1. A directed two-hybrid screen for MTM interactions. (A) Schematic diagram indicating domain structure of the myotubularin family. Point mutations used in this study are indicated by asterisks. (B) Example of a yeast two-hybrid screen for MTMR4 interactions with other MTM family members. Yeast cells were cotransfected with bait-MTMR4 and all different preys (AC). On the selective QDO plate (right), growth (interaction) was observed with MTMR3 and MTMR4 in the prey vectors (see scheme on the left). A catalytically inactive mutant of MTMR3 (C413S) retains binding to MTMR4. As a control, the same co-transformations were plated onto SD-leu/trp (centre).

View larger version (39K): [in a new window]   Fig. 2. Co-immunoprecipitation of human myotubularin family members. HeLa cells were transfected with HAMTMR3, HA-MTMR4 or HAMTMR9, and different EGFP-tagged myotubularins as indicated. EGFP proteins were immunoprecipitated from cell lysates and processed for western blotting. Corresponding membranes were blotted with an anti-HA antibody (top panels) and confirmed interactions identified in the two-hybrid screen. (A) MTMR3 and MTMR4 interact with each other and with themselves. A truncated form of MTMR3 (1-821) lacking the Cterminal coiled-coil domain preserves this interaction. (B) MTMR8 interacts with MTMR9 (arrow, top panel). Membranes were re-probed with anti-EGFP (bottom panels) and lysates with an anti-HA (middle panels) showing the efficiency of EGFP immunoprecipitation and HA-protein expression levels, respectively.

View larger version (71K): [in a new window]   Fig. 3. Subcellular localization of myotubularins in HeLa cells. HeLa cells were transfected for 48 hours with the different EGFP-tagged myotubularins as indicated (A-L). Following fixation, cells were examined by confocal microscopy; representative images are shown. Bars, 10 µm.

View larger version (62K): [in a new window]   Fig. 4. MTMR4 localizes to endosomes. HeLa cells were transfected with EGFP-MTMR4 and co-stained after fixation with anti-EEA1 or anti-Hrs respectively (A,B). (C) AlexaFluor594-Transferrin was internalised into transfected cells during a 30 minute incubation. (D) Transfected cells were treated with wortmannin for 1 hour before fixation. Bar, 10 µm.

View larger version (54K): [in a new window]   Fig. 5. Dephosphorylation of an endosomal pool of PtdIns3P by MTMs. HeLa cells were transfected with MTM1 (A-C), MTMR2 (D-F), MTMR3 (G-I), MTMR4 (J-L), fixed and endosomal PtdIns3P was detected using a biotinylated-GST-2xFYVE protein. Control cells are shown in N and wortmannin-treated cells in M. Bars, 10 µm.

View larger version (61K): [in a new window]   Fig. 6. Localisation and influence upon the endosomal PtdIns3P pool following mutant MTMR4 expression. HeLa cells were transfected with HA-MTMR4-C407S (catalytically inactive, A-C and G-I) or HA-MTMR4-C1169S (FYVE domain mutant, D-F and J-L) fixed and stained with anti-HA and EEA1 antibodies (A-F) or anti-HA and biotinyl-GST-2xFYVE probe (G-L) as described in Materials and Methods. Bar, 10 µm.

View larger version (58K): [in a new window]   Fig. 7. MTMR4 (C407S) inhibits EGF receptor degradation. HeLa cells were transfected with phosphatase-inactive MTMR4, pHAC407S (A-C), or with an additional point mutation in the FYVE domain HA-C407S/C1169S (D-F). 100 ng/ml EGF was added to starved cells for 4 hours, which were then fixed and processed for immunofluorescence of EGFR as described in Materials and Methods. Bars, 10 µm. Cells expressing MTMR4-C407S but not its FYVE domain mutant retain EGFR in numerous bright fluorescent punctae.

View larger version (64K): [in a new window]   Fig. 8. MTMR4 and MTMR3 interaction leads to redistribution. HeLa cells were co-transfected with the following combinations. (A-C) HA-MTMR4 and EGFPMTMR3, (D-F) HA-MTMR4 and EGFP-C2 control vector, (G-I) HA-MTMR10 and EGFP-MTMR4, and (J-L) HAMTMR3-PHG/C413S and EGFP-MTMR4. Merged images are shown on the right. Bar, 10 µm.

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