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Neuron specific Rab4 effector GRASP-1 coordinates membrane specialization and maturation of recycling endosomes - PubMed

  • ️Fri Jan 01 2010

Neuron specific Rab4 effector GRASP-1 coordinates membrane specialization and maturation of recycling endosomes

Casper C Hoogenraad et al. PLoS Biol. 2010.

Erratum in

  • PLoS Biol. 2010;8(9) doi:10.1371/annotation/b17dfb99-8809-4c5a-86c2-c2a0f7ca7f5e.. Sanchez-Martinez, Emma [corrected to Martinez-Sanchez, Emma]

Abstract

The endosomal pathway in neuronal dendrites is essential for membrane receptor trafficking and proper synaptic function and plasticity. However, the molecular mechanisms that organize specific endocytic trafficking routes are poorly understood. Here, we identify GRIP-associated protein-1 (GRASP-1) as a neuron-specific effector of Rab4 and key component of the molecular machinery that coordinates recycling endosome maturation in dendrites. We show that GRASP-1 is necessary for AMPA receptor recycling, maintenance of spine morphology, and synaptic plasticity. At the molecular level, GRASP-1 segregates Rab4 from EEA1/Neep21/Rab5-positive early endosomal membranes and coordinates the coupling to Rab11-labelled recycling endosomes by interacting with the endosomal SNARE syntaxin 13. We propose that GRASP-1 connects early and late recycling endosomal compartments by forming a molecular bridge between Rab-specific membrane domains and the endosomal SNARE machinery. The data uncover a new mechanism to achieve specificity and directionality in neuronal membrane receptor trafficking.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. GRASP-1 is a Rab4GTP-binding protein.

(A) Silver stained gel showing isolation of GSTRab4-GTPγS binding proteins from brain cytosol. Asterisk denotes band from which GRASP-1 was identified. (B) Western blot of samples from (A) probed with GRASP-1 antibody. (C) Binding assay of 35S-labeled GRASP and GSTRab4-GTPγS, or GSTRab4-GDP, and other GTPγS charged GST-Rab proteins. (D) FLAG-tagged Rabs were co-expressed with myc-GRASP-1 in COS-7 cells. Anti-FLAG immunoprecipitates (IP) were analyzed by Western blot with myc antibody. (E) Hela cells were transfected with GFP-Rab4, myc-GRASP-1, or both. Prior to fixation, cells were incubated for 60 min with Alexa594-labeled Tf at 37°C. Bar is 10 µm. (F) Coiled-coil prediction and domain architecture of GRASP-1. Glu, glutamic acid rich domain; asterisk, caspase-3 cleavage site; GRIPBD, GRIP1 binding domain. (G) Binding domain analysis using lysates of COS-7 cells expressing myc-tagged GRASP-1 truncations and GTPγS-charged GST-Rab4.

Figure 2
Figure 2. GRASP-1 does not have GEF activity on H-ras and Rab4.

(A–B) 0.2 µM H-ras or Rab4 loaded with fluorescent mantGDP was incubated with an excess of GDP at 25°C, in the absence or in the presence of 10 µM GRASP-1(1–594), 0.2 µM cdc-25, or 10 µM EDTA. Dissociation of mGDP was monitored by measuring the decrease in relative fluorescence that accompanies release of mGDP from the GTPase. (C) COS-7 cells were transfected with HA-Hras in combination with indicated constructs and treated with or without PMA. Ras-GTP was isolated on GSH beads containing the ras binding domain of the ras effector raf and analyzed by Western blot with HA antibody. Note that full-length GRASP-1 did not increase rasGTP level above non-transfected control. Asterisk and arrowhead in HA Western blot of input material denote a background band and the position of HA-ras, respectively.

Figure 3
Figure 3. Colocalization of GRASP-1 and Rab4 in hippocampal neurons.

(A) Expression pattern of Rab4 and GRASP-1 in mouse tissue and cultured cells visualized on Western blot. (B–F) Representative images of hippocampal neurons double-labeled with antibodies against GRASP-1 and endogenous markers. (B) MAP2 and GRASP-1, arrow denotes axon and arrowheads dendrites. (C) MAP2 and GRASP-1, arrow heads mark GRASP-1 signal beyond the dendritic shaft. (D) PSD-95 and GRASP-1. (E) Bassoon and GRASP-1, arrowheads denote localization of GRASP-1 to synaptic sites. ∼15% of the synapses colocalize with GRASP-1, while the “random” colocalization is ∼5% as determined by rotating the red channel image. (F) Rab4 and GRASP-1 in the cell body (left) and dendrites (right). Arrowheads denote areas of colocalization, inset show magnified regions. Bar in B is 10 µm; Bar in (C–F) is 1 µm. (G) Image of the cell body of hippocampal neurons transfected at DIV13 with GFP-Rab4 and stained for GRASP-1. Magnified region is shown as inset; note the partial localization of GRASP-1 on the distal domain of GFP-Rab4 endosomes. Bar is 1 µm. (H) Simultaneous imaging of GFP-Rab4 (green) and mRFP-GRASP-1 (red) in transfected hippocampal neurons. Successive frames are shown and time (seconds) is indicated in the merge panel.

Figure 4
Figure 4. Endogenous GRASP-1, Rab4, and syntaxin 13 coincide on recycling endosomal tubules.

Immunogold EM of hippocampal neurons labeled with 10 nm protein A gold for Rab4 and with 15 nm protein A gold for GRASP-1 (A), with 10 nm protein A gold for syntaxin 13 and with 15 nm protein A gold for GRASP-1 (B), with 10 nm protein A gold for syntaxin 13 and with 15 nm protein A gold for Rab4 (C), or with 15 nm protein A gold for GRASP-1, with 5 nm protein gold for syntaxin 13, and with 10 nm protein A gold for rab4 (D). Arrow denotes tubular endosomal membrane to which GRASP-1, syntaxin 13, and Rab4 localized. EE indicates early endosomes and scale bar is 100 nm.

Figure 5
Figure 5. GRASP-1 is required for the maintenance of dendritic spines.

(A) Representative high magnification images of dendrites of hippocampal neurons co-transfected at DIV13 for 4 d with β-galactosidase (to mark the dendrites), and either pSuper, pSuper-GRASP-1-shRNA#2, GRASP-1-shRNA#2 and GFP-GRASP-1*, Rab4S22N or Rab11S25N, and labeled with anti-β-galactosidase. (B) Quantification of number of protrusions per 10 µm dendrites in hippocampal neurons transfected as indicated in (A). (C) Percentage of spines of hippocampal neurons transfected as indicated in (A). (D) Neurons expressing GFP (to mark the dendrite), and either pSuper or pSuper-GRASP-1-shRNA#2 were stimulated with glycine (200 mM, 3 min), and then imaged for >30 min after glycine stimulation. Arrows indicated spine formation. Closed and open arrowheads represent spine growth and stable protrusions, respectively. (E) Quantification of protrusion formation (top) and spine growth (bottom) following glycine stimulation. N, number of dendritic protrusions per 10 µm at the indicated time; N0, average number of dendritic protrusions per 10 µm before application of glycine. Spine growth was probed as the change in sum of spine widths per 10 µm and comprises both addition of new spines and growth of pre-existing spines. Glycine-stimulated spine growth is blocked by GRASP-1-shRNA#2 (bottom). (F) High magnification images of dendrites of hippocampal neurons cotransfected at DIV13 for 4 d with myc-GRASP-1 (red) and GFP-TfR. (G,H) Percentage of spines containing TfR-GFP positive endosomes at the indicated locations. Hippocampal neurons were co-transfected at DIV13 for 4 d with β-galactosidase (to mark dendrites) and GFP-TfR (to mark endosomes) and pSuper control vector or pSuper-GRASP-1-shRNA#2 as shown in (H). Closed and open arrowheads denote protrusions with and without GFP-TfR marked endosomes in the spine head, respectively. Error bars indicate S.E.M. ** p<0.005. *** p<0.0005. Bar is 1 µm.

Figure 6
Figure 6. Knock-down of GRASP-1 reduces AMPAR recycling.

(A) Representative merge image of surface HA-GluR2 (blue) and internalized HA-GluR2 (green) in soma and dendrites of hippocampal neurons labeled for GRASP-1 (red) after 10 min AMPA stimulation. Bar is 10 µm. (B,C) Quantification of the surface fluorescence intensities of endogenous GluR1 (B) and GluR2 (C) in control pSuper vector or GRASP-1-shRNA#2 transfected neurons. The cells were untreated (0 min) or stimulated with AMPA for indicated times. Histograms show fluorescent intensity of surface GluR subunit staining relative to the intensity of GFP transfected control neurons at basal levels. n = 20 cells for each group. (D,E) Representative images of hippocampal neurons stained for endogenous surface GluR1 (D) and GluR2 (E). Hippocampal neurons at DIV13 were cotransfected with GFP and pSuper control vector or GRASP-1-shRNA#2. At DIV17, neurons were fixed (0 min, no treatment) or stimulated for 2 min with 100 µM AMPA in the presence of 50 µM APV and further incubated for a total of 10 or 60 min before fixation. Endogenous surface GluR1 (D) or GluR2 (E) was revealed by immunofluorescence labeling without permeabilization using specific extracellular AMPAR antibodies. Bar is 20 µm. (F) Neurons transfected with GFP, HA-GluR2, and either pSuper control vector or GRASP-1-shRNA#2 were stained live with an anti-HA antibody, stimulated for 2 min with AMPA/APV, acid stripped, and incubated in conditioned media for 45 min. Recycled HA-GluR2 (blue) and internalized HA-GluR2 (red) were sequentially labeled. Bar is 1 µm. (G) Quantification of the ratio of recycled to internalized HA-GluR2 and normalized to unstimulated wild-type control neurons (HA-GluR2 recycling index) as indicated in (F). Error bars indicate S.E.M. * p<0.05. ** p<0.005. *** p<0.0005.

Figure 7
Figure 7. Effect of GRASP-1 knock-down on synaptic transmission and plasticity in hippocampal slice.

(A,B) AMPA and NMDA receptor-mediated excitatory synaptic responses were measured from neurons transfected with Luciferase-shRNA (A, control) and GRASP-1-shRNA#5 (B). Top, sample traces mediated by AMPAR (downward) and NMDAR (upward) from pairs of shRNA transfected (Luciferase or GRASP-1-shRNA#5) and neighboring untransfected (Untrans) neurons. Stimulus artifacts were truncated from the traces. Bottom, summary graphs of EPSC amplitudes (AMPA-R-EPSCs and NMDA-R-EPSCs) from shRNA transfected and neighboring untransfected cells. Number of cell pairs: Luciferase-shRNA, 18 and 10; GRASP-1-shRNA#5, 15 and 8 for AMPA and NMDAR-EPSC. NS, not significant. Error bars indicate S.E.M. (C,D) LTP was induced in shRNAs expressing and neighboring untransfected cells by pairing depolarization to 0 mV with 2 Hz stimulation for 100s. Left, sample AMPAR-EPSC traces from untransfected and Luciferase or GRASP-1 shRNA transfected neurons. Currents before (black) and after (gray) are superimposed. Right, time course of AMPA-EPSCs after LTP induction (LTP was induced at t = 0). The time points at which sample traces were obtained are indicated by 1 and 2. Number of cell pairs: Luciferase-shRNA, 6; GRASP-1-shRNA#5, 8. * p<0.05.

Figure 8
Figure 8. GRASP-1 segregates Rab4 from EEA1 positive endosomal membranes.

(A) Representative images of dendrites of hippocampal neurons cotransfected at DIV13 for 4 d with myc-GRASP-1 (red) and either GFP-Rab4 (upper row) or GFP-Rab5 (bottom row). (B) Percentage of colocalization between myc-GRASP-1 and Rab proteins in neurons as indicated in (A). (C) Percentage of Rab4 and EEA1 colocalization in cell body and dendrites as indicated in (E). Error bars indicate S.E.M. *** p<0.0005. (D) Representative images of dendrites of hippocampal neurons double-labeled with anti-GRASP-1 (red) and anti-EEA1 (green) antibodies. (E) Representative images of dendrites of hippocampal neurons cotransfected at DIV13 for 4 d with GFP-Rab4 and pSuper control vector, myc-GRASP-1, or pSuper-GRASP-1-shRNA#2 and labeled with anti-EEA1 (red) and anti-myc (blue) antibodies. Bar is 1 µm.

Figure 9
Figure 9. GRASP-1 couples Rab4 and Rab11 domains.

(A) Representative images of dendrites of hippocampal neurons cotransfected at DIV13 for 4 d with myc-GRASP-1 and either GFP-tagged Rab7 or Rab11 and labeled with anti-myc (red). Bar is 1 µm. (B) Simultaneous imaging of GFP-Rab11 (green) and mRFP-GRASP-1 (red) in transfected hippocampal neurons. Successive frames are shown and time (seconds) is indicated in the merge panel. (C) Percentage of colocalization between myc-GRASP-1 and Rab proteins in neurons as indicated in (A). Error bars indicate S.E.M. *** p<0.0005. (D) Percentage of colocalization between Rab4 and Rab11 domains in neurons co-transfected with GFP-Rab4 and HA-Rab11 with either myc-GRASP-1, pSuper-GRASP-1-shRNA#2, or pSuper-GRASP-1-shRNA#2 and GFP-GRASP-1* as indicated in (F). (E) Images of cell body of hippocampal neurons triple transfected at DIV13 for 4 d with GFP-Rab4, HA-Rab11, and myc-GRASP-1 and labeled with anti-HA (red) or anti-myc (blue) antibodies. Bar is 10 µm. (F) Representative images of dendrites of hippocampal neurons cotransfected at DIV13 for 4 d with GFP-Rab4 and HA-Rab11 and pSuper control vector, myc-GRASP-1, or pSuper-GRASP-1-shRNA#2 and labeled with anti-HA (red) or anti-myc (inset) antibodies. Bar is 1 µm.

Figure 10
Figure 10. Syntaxin 13 interacts with the C-terminal domain of GRASP-1.

(A) Lysates of COS-7 cells cotransfected with GFP-GRASP-1 and myc-syntaxins were immunoprecipitated with anti-GFP antibody and analyzed by Western blot. (B) Lysates of COS-7 cells cotransfected with GFP-syntaxin 13 and full-length myc-GRASP-1 (1–837) or truncated myc-GRASP-1 constructs (1–695 or 695–837) were immunoprecipitated with anti-GFP antibody and analyzed by Western blot. Asterisk indicates background band. Arrows point to co-precipitated GRASP-1 proteins. (C) Binding assay using lysates of COS-7 cells expressing myc-syntaxin 13 with or without GFP-GRASP-1 and GMP-PNP-charged GST-rab4. Note that myc-syntaxin 13 is only isolated on the beads in the presence of GRASP-1. (D) Binding assay using lysate of COS-7 cells transfected with GFP-GRASP-1(594–837) and GST-syntaxins without transmembrane domain (ΔTM). GRASP-1 was analyzed by Western blot with antibody against GFP. (E) Binding assay of 35S-labeled GRASP-1 and immobilized GST-syntaxin 13ΔTM.

Figure 11
Figure 11. Syntaxin 13 coincides with GRASP-1 and segregates Rab4/Rab11 domains.

(A) Representative image of hippocampal neuron triple transfected at DIV13 for 4 d with GFP-Rab4, HA-GRASP-1, and myc-syntaxin 13 and labeled with anti-HA (blue) or anti-myc (red) antibodies. Magnified region of the cell body is shown to indicate the strong colocalization of GRASP-1, Rab4, and syntaxin 13. (B) Representative images of dendrites of hippocampal neurons transfected at DIV13 with GFP-GRASP-1 for 4 d and labeled with anti-syntaxin 13 (red). (C) Representative images of dendrites of hippocampal neurons transfected at DIV13 with GFP-GRASP-1 for 4 d and labeled with anti-Neep21 (red). (D) Representative images of dendrites of hippocampal neurons cotransfected at DIV13 for 4 d with myc-syntaxin 13 and control vector or HA-GRASP-1 and labeled with anti-myc (green), anti-HA (blue), and anti-Neep21 (red). (E) Representative images of dendrites of hippocampal neurons cotransfected at DIV13 for 4 d with GFP-Rab4, HA-Rab11, and control vector or myc-syntaxin 13ΔTM and labeled with anti-myc (blue) and anti-HA (red). (F) Percentage of colocalization between HA-GRASP-1 and myc-syntaxin 1 or myc-syntaxin 13 in neurons. (G) Percentage of colocalization between myc-syntaxin 13 and Neep21 in dendrites as indicated in (D). (H) Percentage of colocalization between GFP-Rab4 and HA-Rab11 domains in dendrites expressing myc-syntaxin 13ΔTM as indicated in (E). Error bars indicate S.E.M. ** p<0.005. *** p<0.0005. Bar in A is 10 µm; Bar in (B–E) is 1 µm.

Figure 12
Figure 12. Model for the role of GRASP-1 in endosome recycling.

Endosomes can be viewed as mosaic distribution of Rab4, Rab5, and Rab11 domains that dynamically interact via effector proteins and SNAREs. The Rab5 domain allows entry into the early/sorting endosome, whereas the Rab4 and Rab11 domains contain the machinery that is necessary for sorting and recycling membranes and receptors back to the plasma membrane. (A) GRASP-1 binds to Rab4 and syntaxin 13 and couples Rab4 and Rab11 recycling endosomes. The complex formed between GRASP-1 and t-SNARE syntaxin 13 might mediate fusion between Rab4 and Rab11 endosomes. (B) Absence of GRASP-1 interferes with complex formation at the recycling step, causing cargo accumulation in early endosomes, impairment of receptor expression, and changes in spine morphology. (C) Overexpression of GRASP-1 leads to recruitment of syntaxin 13 and strongly couples Rab4 and Rab11 domains, causing accumulation of internalized receptors in recycling endosomes. Consistent with the observed decrease in AMPAR clusters , Caspase-3 cleavage of GRASP-1 might separate the N-terminal Rab4 domain from the C-terminal syntaxin 13 binding site and disrupt the coupling between Rab4 and Rab11 domains.

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