Protrudin Induces Neurite Formation by Directional Membrane Trafficking

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Science  03 Nov 2006:
Vol. 314, Issue 5800, pp. 818-821
DOI: 10.1126/science.1134027


Guanosine triphosphatases of the Rab family are key regulators of membrane trafficking, with Rab11 playing a specific role in membrane recycling. We identified a mammalian protein, protrudin, that promoted neurite formation through interaction with the guanosine diphosphate (GDP)–bound form of Rab11. Phosphorylation of protrudin by extracellular signal–regulated kinase (ERK) in response to nerve growth factor promoted protrudin association with Rab11-GDP. Down-regulation of protrudin by RNA interference induced membrane extension in all directions and inhibited neurite formation. Thus, protrudin regulates Rab11-dependent membrane recycling to promote the directional membrane trafficking required for neurite formation.

The molecular mechanisms that underlie neurite formation include both cytoskeletal remodeling and membrane trafficking (1, 2). During this process, membrane components are transported in a directional manner within the cell by a membrane recycling system, resulting in expansion of the cell surface area in the region of neurite formation (3, 4). Rab11 regulates membrane recycling back to the plasma membrane and constitutive exocytosis (514).

We isolated protrudin as a protein that interacts with FKBP38, a multifunctional membranous chaperone (15). Protrudin contains a Rab11 binding domain (RBD11), a guanosine diphosphate (GDP) dissociation inhibitor (GDI) consensus sequence (16), two hydrophobic domains (HP-1 and HP-2), an FFAT motif (an endoplasmic reticulum–targeting signal) (17), a coiled-coil domain, and a FYVE domain (a phosphoinositide binding domain) (fig. S1A) (18). These characteristics suggested that protrudin might function in membrane trafficking, especially in membrane recycling.

Expression of FLAG-protrudin in HeLa cells resulted in the generation of long processes (Fig. 1A). Processes were observed in 5 to 30% of transfected cells. We also monitored process formation by time-lapse video microscopy in living HeLa cells expressing enhanced green fluorescent protein (EGFP)–protrudin (Fig. 1B and movie S1). The processes appeared to grow in a manner similar to that of neurites with ruffling lamellipodia. In rat hippocampal neurons, expression of the exogenous protrudin promoted neurite extension in the neurons (Fig. 1C and fig. S2). We next analyzed the effects of depletion of endogenous protrudin by RNA interference (RNAi) in the rat pheochromocytoma PC12 cell line, which extends neurites in response to nerve growth factor (NGF) (Fig. 1D). Depletion of protrudin resulted in inhibition of NGF-induced neurite outgrowth and triggered spreading of the plasma membrane in all directions (Fig. 1, E and F). Thus, protrudin is necessary for neurite formation and may function by regulating the direction of membrane sorting.

Fig. 1.

Protrudin induces outgrowth of cellular projections. (A) HeLa cells were transfected with a FLAG-protrudin vector and stained with anti-FLAG (green), phalloidin (red), and Hoechst 33258 (WAKO, Osaka, Japan) (blue). (B) HeLa cells were transfected with EGFP-protrudin, and the fluorescence was monitored by video (movie S1). (C) Rat hippocampal neurons were transfected with FLAG-protrudin or the empty vector and stained with anti–βIII-tubulin. (D) PC12 cells were transfected with short hairpin RNA (shRNA) vectors specific for protrudin mRNA (KD-2, -3, -4, and -5) or control (SC-3 and -5) and subjected to immunoblotting with anti-protrudin and anti–heat shock protein 90 (anti-Hsp90). (E) PC12 cells were transfected with control (SC-3) or protrudin (KD-3) shRNA vectors, stimulated with NGF, and stained with anti–α-tubulin (green) and phalloidin (red). (F) PC12 cells were transfected with a vector encoding Venus and either control (SC-3) or protrudin (KD-3) shRNA, stimulated with NGF, and examined for Venus fluorescence.

In mouse, protrudin was most abundant in the cerebrum and the cerebellum (Fig. 2A). In mouse primary neurons in the early stage of differentiation, protrudin was localized to the pericentrosomal region and neurites (Fig. 2B). At later stages, it was detected in both dendrites and axons and was concentrated at the growth cones (Fig. 2C). We next examined the effect of NGF on the subcellular distribution of protrudin in PC12 cells (Fig. 2D). In the absence of NGF, protrudin was distributed diffusely throughout the cell body. In cells exposed to NGF for 6 hours, protrudin was markedly concentrated in the pericentrosomal region, where it colocalized with the recycling endosome marker Rab11 (fig. S3, A to C). After culture with NGF for 24 hours, protrudin was distributed from the pericentrosomal region to the tips of neurites (Fig. 2D). The overall abundance of protrudin was not affected by NGF (fig. S3D). Immuno–electron microscopic analysis revealed that protrudin appears to be associated with vesicle membranes (fig. S3E). Thus, protrudin undergoes a dynamic redistribution during neuritogenesis.

Fig. 2.

Subcellular redistribution of protrudin during neuritogenesis. (A) Tissue distribution of protrudin in mice. (B and C) Cells isolated from embryonic mouse cerebral cortex were cultured for 1 (B) or 3 (C) days and stained with anti-protrudin (green). Arrowhead in (B) indicates the pericentrosomal region. The boxed region in (C) is shown at higher magnification in the bottom image; the arrowhead indicates the growth cone. (D) PC12 cells were incubated with NGF and stained with anti-protrudin (top, green). Differential interference contrast (DIC) images are shown in the middle, and the boxed regions are shown at bottom merged with protrudin.

Although protrudin contains a consensus sequence for an RBD11, the amino acids required for interaction with the GTP-bound form of Rab11 are not conserved in this domain of protrudin (19) (Fig. 3A). The amino acid sequence of the RBD11 of protrudin is similar to the corresponding sequences of GDI-α and GDI-β, both of which interact with the GDP-bound form of many Rab proteins (20, 21). We thus examined whether protrudin actually interacted with Rab11. Endogenous protrudin did indeed interact with endogenous Rab11 in PC12 cells, and this interaction was enhanced by NGF treatment (Fig. 3B). FLAG-protrudin also interacted with hemagglutinin (HA)–tagged Rab11 in human embryonic kidney (HEK293T) cells (Fig. 3C). Protrudin preferentially interacted with the GDP-bound form of Rab11 [as represented by the GTP binding–deficient mutant Ser25→Asn25 (S25N)] rather than with the GTP-bound form [as represented by the GTPase-deficient mutant Gln70→Leu70 (Q70L)] (22, 23) (Fig. 3D). The RBD11 of the Rab11 effector FIP2 interacted specifically with Rab11-GTP, whereas the RBD11 of protrudin specifically interacted with Rab11-GDP (fig. S4A). Reciprocal co-immunoprecipitation analysis as well as an in vitro pull-down assay confirmed that protrudin preferentially interacts with Rab11-GDP (fig. S4, B and C). We also generated a series of deletion mutants of protrudin (fig. S1B) and tested them for the ability to bind Rab11. Rab11(S25N) interacted with the N1 mutant but not with the ΔRBD-N1 mutant (fig. S4D). Furthermore, a protrudin deletion mutant lacking the RBD11 (ΔRBD in fig. S1B) could not restore neurite formation in PC12 cells depleted of protrudin by RNAi (fig. S5A) or induce process formation in HeLa cells (fig. S5B). Thus, protrudin preferentially binds to Rab11-GDP, and this association is required for neurite formation.

Fig. 3.

Interaction of protrudin with Rab11. (A) Alignment of the amino acid sequence (17) of the RBD11 of protrudin with the Rab11 effector proteins and GDIs. Conserved residues are shaded in red or blue. Asterisks denote critical residues for interaction with Rab11-GTP (shaded in green). (B) Immunoprecipitation (IP) of PC12 lysates with anti-protrudin and immunoblotting (IB) with anti-Rab11 and -protrudin. (C) FLAG-protrudin expressed with HA-Rab4, -Rab5, or -Rab11 in HEK293T cells was immunoprecipitated and subjected to IB with anti-HA and -FLAG. (D) FLAG-protrudin expressed with either wild-type (WT) or mutant (S25N or Q70L) HA-Rab11 in HEK293T cells was immunoprecipitated and subjected to IB with anti-HA and -FLAG. (E) PC12 cells expressing HA-protrudin were metabolically labeled with [32P]orthophosphate in the absence or presence of NGF (top). Alternatively, PC12 cells expressing HA-protrudin and Myc-MEK1 (SDSE or K97S) were similarly labeled (bottom). Cell lysates were subjected to IP with anti-HA and to autoradiography or to IB with anti-HA. (F) PC12 cells expressing HA-protrudin as well as Myc-MEK1 (SDSE or K97S) were incubated with or without NGF. Cell lysates were subjected to IP with anti-HA and to 2D-PAGE followed by IB with anti-HA. (G) HEK293T cells transfected with FLAG-protrudin, HA-Rab11 (WT or S25N), and Myc-MEK1 (SDSE or K97S) were subjected to IP with anti-FLAG and to IB with anti-FLAG, -HA, -Myc, -ERK, and -phospho-ERK(p-ERK). (H) HEK293T cells transfected with FLAG-protrudin (WT, D-mut, or P-mut-1), Myc-MEK1(SDSE), and HA-Rab11(S25N) were subjected to IP with anti-FLAG and to IB with anti-HA, -FLAG, and -Myc.

Sustained activation of the mitogen-activated protein kinase (MAPK) ERK accompanies NGF-dependent neurite extension in PC12 cells (24) (fig. S6A). PC12 cells expressing a constitutively active form of MEK1 [MEK1(SDSE)], which activated ERK, manifested neurite formation in the absence of NGF (fig. S6, B and C). Protrudin and MEK1(SDSE) showed a synergistic effect on process formation in HeLa cells, whereas a kinase-negative form of MEK1 [MEK1(K97S)] antagonized the process-forming activity of protrudin (fig. S6D). Protrudin contains six potential ERK phosphorylation sites as well as two consensus ERK binding (ERK D) domains (fig. S1C). Phosphorylation of protrudin was increased by NGF treatment or MEK1(SDSE) expression, but not by MEK1(K97S) expression, in PC12 cells (Fig. 3E). Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) also suggested that protrudin is phosphorylated by ERK in response to NGF (Fig. 3F). MEK1(K97S) expression inhibited the NGF-induced shift, suggesting that ERK activation is essential for the phosphorylation of protrudin elicited by NGF.

We examined the effect of ERK activation on protrudin-Rab11 interaction in HEK293T cells. Expression of MEK1(SDSE) enhanced the interaction between protrudin and Rab11(S25N) (Fig. 3G). At high amounts of MEK1(SDSE) expression, both wild-type and S25N forms of Rab11 interacted substantially with protrudin (fig. S7, A and B). Furthermore, protrudin mutants that lack some of potential ERK phosphorylation sites (P-mut-1 and -4) or both intact ERK D domains (D-mut) (fig. S1C) showed markedly reduced affinities for Rab11 compared with the affinities of wild-type protrudin (Fig. 3H and fig. S7C). Replacement of potential ERK phosphorylation sites in other combinations (P-mut-2 and -3) did not affect the binding (fig. S7D). Thus, phosphorylation of protrudin at multiple sites in response to NGF-ERK signaling promotes its interaction with Rab11.

We next investigated the effect of Rab11 on the morphology of PC12 cells. The morphology of cells expressing Rab11(Q70L) appeared nearly identical to that of cells depleted of endogenous protrudin (Fig. 4A). Conversely, the phenotype conferred by expression of Rab11(S25N) was similar to that conferred by expression of protrudin. These experiments were combined to examine the genetic relation between protrudin and Rab11. Expression of Rab11(S25N) induced neurite formation in cells depleted of endogenous protrudin (Fig. 4B), whereas overexpression of protrudin had no effect on the morphology of cells expressing Rab11(Q70L) (Fig. 4C). In addition, expression of Rab11(Q70L) inhibited process formation induced by protrudin in HeLa cells (movie S2). Thus, protrudin is indeed an upstream inhibitor of Rab11 function.

Fig. 4.

Protrudin induces directional membrane extension through regulation of vesicular traffic. (A) PC12 cells expressing HA-Rab11 (S25N or Q70L) or HA-Rab4 (S22N or Q67L) were stimulated with NGF and stained with anti-HA (green) and phalloidin (red). (B) PC12 cells transfected with a vector encoding both protrudin shRNA and Venus (green) were also transfected with a HA-Rab11(S25N) vector, incubated with NGF, and stained with anti-HA (red). Arrows indicate cells expressing both Venus (protrudin shRNA) and HA-Rab11(S25N). (C) PC12 cells were transfected with HA-Rab11(Q70L) and FLAG-protrudin, stimulated with NGF, and stained with anti-HA (red) and anti-FLAG (green). (D) PC12 cells transiently infected with a retrovirus encoding chicken NgCAM were transfected with control or protrudin shRNAs or with Rab11 (Q70L). The surface expression of NgCAM was examined by immunostaining with anti-NgCAM (green) without cell permeabilization. Arrowheads indicate the prominent distribution of NgCAM on a neurite.

To investigate the role of the protrudin-Rab11 system in directed membrane traffic, we observed the transport of NgCAM, a cell adhesion molecule that is delivered to the somatodendritic plasma membrane and then transported to the axonal plasma membrane via recycling endosomes (2527). Chicken NgCAM expressed in PC12 cells initially accumulated, presumably in the endoplasmic reticulum–Golgi compartment, and was subsequently transported to the plasma membrane of some neurites, but not to that of the soma, in the presence of NGF (Fig. 4D and fig. S8). In contrast, cells depleted of protrudin by RNAi or those expressing Rab11(Q70L) exhibited prominent surface expression of NgCAM on the soma (Fig. 4D). Thus, protrudin is essential for directional membrane trafficking to neurites.

Our data indicate that the protrudin-Rab11 system is an important determinant of the direction of membrane trafficking and neurite formation. It was recently shown that ZFYVE27 (a synonym of protrudin) is mutated in a German family with an autosomal dominant form of hereditary spastic paraplegia (AD-HSP), which is characterized by selective degeneration of axons (28). The phenotype of the affected individuals was similar to that of patients with AD-HSP caused by mutation of spastin, a protein implicated in neuronal vesicular cargo trafficking. Protrudin is thought to interact with spastin via its FYVE domain in the COOH-terminal region of the protein. This genetic evidence supports our conclusion that protrudin plays a central role in membrane trafficking in neurons. Rab11, protrudin, and spastin may together constitute a system for the regulation of vesicular transport in neurons, and impairment of this system may be responsible for the pathogenesis of AD-HSP.

Supporting Online Material

Materials and Methods

Figs. S1 to S8

Movies S1 and S2

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