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• W2022997685 abstract "Article10 May 2007free access G protein βγ subunit interaction with the dynein light-chain component Tctex-1 regulates neurite outgrowth Pallavi Sachdev Pallavi Sachdev Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, NY, USA Search for more papers by this author Santosh Menon Santosh Menon Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, NY, USA Search for more papers by this author David B Kastner David B Kastner Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, NY, USAPresent address: Stanford University Medical School, Palo Alto, CA 94305, USA Search for more papers by this author Jen-Zen Chuang Jen-Zen Chuang Department of Ophthalmology, Weill Medical College of Cornell University, New York, NY, USA Search for more papers by this author Ting-Yu Yeh Ting-Yu Yeh Department of Ophthalmology, Weill Medical College of Cornell University, New York, NY, USAPresent address: Johns Hopkins University, Baltimore, MD 21218, USA Search for more papers by this author Cecilia Conde Cecilia Conde INIMEC-CONICET, Cordoba, Argentina Search for more papers by this author Alfredo Caceres Alfredo Caceres INIMEC-CONICET, Cordoba, Argentina Search for more papers by this author Ching-Hwa Sung Ching-Hwa Sung Department of Ophthalmology, Weill Medical College of Cornell University, New York, NY, USA Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, NY, USA Search for more papers by this author Thomas P Sakmar Corresponding Author Thomas P Sakmar Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, NY, USA Search for more papers by this author Pallavi Sachdev Pallavi Sachdev Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, NY, USA Search for more papers by this author Santosh Menon Santosh Menon Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, NY, USA Search for more papers by this author David B Kastner David B Kastner Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, NY, USAPresent address: Stanford University Medical School, Palo Alto, CA 94305, USA Search for more papers by this author Jen-Zen Chuang Jen-Zen Chuang Department of Ophthalmology, Weill Medical College of Cornell University, New York, NY, USA Search for more papers by this author Ting-Yu Yeh Ting-Yu Yeh Department of Ophthalmology, Weill Medical College of Cornell University, New York, NY, USAPresent address: Johns Hopkins University, Baltimore, MD 21218, USA Search for more papers by this author Cecilia Conde Cecilia Conde INIMEC-CONICET, Cordoba, Argentina Search for more papers by this author Alfredo Caceres Alfredo Caceres INIMEC-CONICET, Cordoba, Argentina Search for more papers by this author Ching-Hwa Sung Ching-Hwa Sung Department of Ophthalmology, Weill Medical College of Cornell University, New York, NY, USA Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, NY, USA Search for more papers by this author Thomas P Sakmar Corresponding Author Thomas P Sakmar Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, NY, USA Search for more papers by this author Author Information Pallavi Sachdev1, Santosh Menon1, David B Kastner1, Jen-Zen Chuang2, Ting-Yu Yeh2, Cecilia Conde3, Alfredo Caceres3, Ching-Hwa Sung2,4 and Thomas P Sakmar 1 1Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, NY, USA 2Department of Ophthalmology, Weill Medical College of Cornell University, New York, NY, USA 3INIMEC-CONICET, Cordoba, Argentina 4Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, NY, USA *Corresponding author. Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, 1230 York Avenue, Box 187, New York City, NY 10021, USA. Tel.: +1 212 327 8288; Fax: +1 212 327 7904; E-mail: [email protected] The EMBO Journal (2007)26:2621-2632https://doi.org/10.1038/sj.emboj.7601716 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Tctex-1, a light-chain component of the cytoplasmic dynein motor complex, can function independently of dynein to regulate multiple steps in neuronal development. However, how dynein-associated and dynein-free pools of Tctex-1 are maintained in the cell is not known. Tctex-1 was recently identified as a Gβγ-binding protein and shown to be identical to the receptor-independent activator of G protein signaling AGS2. We propose a novel role for the interaction of Gβγ with Tctex-1 in neurite outgrowth. Ectopic expression of either Tctex-1 or Gβγ promotes neurite outgrowth whereas interfering with their function inhibits neuritogenesis. Using embryonic mouse brain extracts, we demonstrate an endogenous Gβγ–Tctex-1 complex and show that Gβγ co-segregates with dynein-free fractions of Tctex-1. Furthermore, Gβ competes with the dynein intermediate chain for binding to Tctex-1, regulating assembly of Tctex-1 into the dynein motor complex. We propose that Tctex-1 is a novel effector of Gβγ, and that Gβγ–Tctex-1 complex plays a key role in the dynein-independent function of Tctex-1 in regulating neurite outgrowth in primary hippocampal neurons, most likely by modulating actin and microtubule dynamics. Introduction Signaling pathways that lead to neurite outgrowth and the establishment of neuronal polarity remain poorly understood. However, dynamic rearrangements of microtubules and actin filaments at the tips of growing axons are observed during neurite sprouting and elongation, suggesting that molecules that coordinate microtubule and actin microfilament dynamics play key roles in neurite extension and neuronal polarity determination (Fukata et al, 2002; Baas and Buster, 2004). Cytoplasmic dynein light-chain component, Tctex-1, was demonstrated recently to play key roles in initial neurite sprouting, axonal specification and elongation of hippocampal neurons in culture (Chuang et al, 2001, 2005). Cytoplasmic dynein is a microtubule-based, minus-end-directed motor complex involved in various cellular activities, including retrograde trafficking in neurons, Golgi maintenance, breakdown of the nuclear envelope and mitosis (Hirokawa, 1998; Sakato and King, 2004). Dynein comprises two ∼530 kDa heavy chains (DHCs) with ATPase and motor activities, two or three 74-kDa intermediate chains (DICs), and a group of accessory polypeptides including light intermediate chains and light chains (DLCs) (Vallee et al, 2004). DICs link accessory proteins, including the DLCs and the dynactin complex, to the DHC (Waterman-Storer et al, 1995; King, 2000). Three distinct DLC families have been identified: Tctex-1 (DYNTL1; Pfister et al, 2005), LC8 and LC7/Roadblock (Vallee et al, 2004). Several lines of evidence suggest that Tctex-1 might function independently from dynein. First, there is strong biochemical evidence that a dynein-free pool of Tctex-1 exists independently of the dynein complex-associated Tctex-1 (Tai et al, 1998). Secondly, Tctex-1 is abundantly expressed in postmitotic, young neurons and was demonstrated recently to play a key role in neuritogenesis in hippocampal neurons in culture (Chuang et al, 2001, 2005). Cultured hippocampal neurons develop multiple abnormally long neurites when Tctex-1 is overexpressed and fail to develop neurites when Tctex-1 is suppressed (Chuang et al, 2005). The function of Tctex-1 in neuritogenesis was demonstrated to be dynein independent, since a mutant of Tctex-1 (Tctex-1 T94E), which failed to bind to DIC and therefore could not get incorporated into the dynein complex, induced a similar phenotype as the wild-type Tctex-1 protein (Chuang et al, 2005). Finally, Tctex-1 interacts with proteins besides DIC, including rhodopsin (Tai et al, 1999), parathyroid hormone receptor (PTHR) (Sugai et al, 2003), poliovirus receptor CD155 (Ohka et al, 2004), Herpes virus capsid protein VP26 (Douglas et al, 2004), bone morphogenetic receptor type II (BMPR-II) (Machado et al, 2003), the voltage-dependent anion channel (VDAC) (Schwarzer et al, 2002), Fyn kinase (Kai et al, 1997; Mou et al, 1998) and Trk neurotrophin receptor (Yano et al, 2001; Yano and Chao, 2004). How dynein-associated and dynein-free pools of Tctex-1 are maintained in the cell and how the assembly of Tctex-1 into the dynein complex is regulated is not known. Tctex-1 was independently identified as an activator of G protein signaling 2 (AGS2), a receptor-independent activator of heterotrimeric guanine-nucleotide-binding regulatory proteins (G proteins), in a functional yeast screen in yeast (Takesono et al, 1999). AGS molecules can generally be divided into three subgroups: those that directly activate Gα, those that modulate Gα–Gβγ interaction by binding to Gα, and those that modulate Gα–Gβγ interaction by binding to Gβγ (Lanier, 2004). These AGS molecules have since demonstrated novel, non-canonical roles for G protein subunits in cell development and differentiation. For example, silencing of AGS3, a receptor-independent activator of Gβγ signaling, resulted in defects in mitotic spindle orientation and cleavage plane determination of neural progenitors in the developing neocortex demonstrating a novel role for G proteins in regulating neuronal cell fate (Sanada and Tsai, 2005). AGS2/Tctex-1 was reported to bind Gβγ, but the molecular mechanism and functional consequences of the putative Tctex-1–Gβγ interaction have remained unknown (Takesono et al, 1999). Here, we studied the role of Gβγ in regulating the dynein-independent function of Tctex-1 in neuritogenesis. An endogenous Gβγ–Tctex-1 complex can be isolated from embryonic brain lysates and Gβ overlaps with the ‘dynein-free’ Tctex-1 in cell fractionation experiments. Subcellular distribution of Gβγ and Tctex-1 overlap in the cell bodies as well as the growth cones of nascent axons in stage 3 primary cultured hippocampal neurons. Both Tctex-1 and Gβγ overexpression elicit similar phenotypes in primary hippocampal neurons. Interfering with Gβγ function inhibits neuritogenesis and diminishes the ability of Tctex-1 to induce neurite outgrowth. All known Gβ isoforms contain an identical consensus Tctex-1-binding motif first described in DIC (Mok et al, 2001). We show that full-length Gβ1, as well as a Gβ-peptide corresponding to the Tctex-1-binding region of Gβ1, compete with DIC for Tctex-1 binding. We propose that Gβγ binds to Tctex-1 to regulate the dynein-independent pool of Tctex-1 and its incorporation into the dynein motor complex. Finally, we demonstrate that Gβγ–Tctex-1 complex plays a key role in neuritogenesis in an established model of hippocampal neuron differentiation. Results Characterization of Gβγ–Tctex-1 interaction Tctex-1 was shown to interact specifically with Gβγ and not with the Gα subunit of heterotrimeric G proteins (Takesono et al, 1999). We confirmed the interaction of Gβγ with Tctex-1 using purified components. We generated GST-tagged Tctex-1 and purified visual G protein transducin, Gtαβγ. GST–Tctex-1 was incubated with Gtαβγ and the bound Gtβγ subunit was detected using an anti-Gβ antibody. As shown in Figure 1A, GST–Tctex-1 but not GST alone, specifically bound Gtβγ. We also studied the Gβγ–Tctex-1 interaction in a mammalian cell expression system. We cotransfected human embryonic kidney (HEK) cells with Tctex-1 and Gβ1 expression plasmids. Confirming the in vitro data, we showed that Tctex-1 could robustly co-immunoprecipitate (co-IP) with Gβγ (Figure 1B). Tctex-1 did not co-IP with overexpressed Gtα in HEK cells (data not shown). The Gβ subunit consists of six different isoforms, including Gβ1 through 5 and a splice variant of Gβ5 called Gβ5L (long form). Gβ1–4 are about 80% identical to each other, whereas Gβ5 is only about 50% identical to the others. As shown in Figure 1C, all FLAG-tagged Gβ subunits (Gβ1, 2, 3, 5 and 5L) co-immunoprecipitated with Tctex-1 using an anti-Tctex-1 antibody. The expression of Gγ1 or Gγ2 together with Gβ1 did not affect the ability of Tctex-1 to associate with Gβ1. Gγ1 and Gγ2 expression was confirmed by stripping and reprobing the membranes with anti-Gγ1 and anti-Gγ2 antibodies, respectively (data not shown). Even though both Gβ and Tctex-1 are expressed in HEK 293 cells, we failed to co-IP a native endogenous Gβγ–Tctex-1 complex from these cells. Since Tctex-1 is abundantly expressed in postmitotic young neurons and several Gβγ combinations are found in brain, we employed embryonic mouse brain lysates in pull-down assays (Chuang et al, 2001). As shown in Figure 1C, we were able to pull down Gβ along with Tctex-1, demonstrating a native endogenous Gβγ–Tctex-1 complex in mouse embryonic brain lysate. Figure 1.Gβγ interacts with Tctex-1. (A) Transducin Gαtβ1γ1 (Gt) interacts with GST-fused Tctex-1. GST alone (lanes 1–3) or GST-fused Tctex-1 (lanes 4–6) (300 nM) were incubated with Gt (40 nM) (lanes 1 and 4), plus 10 μM GDP (lanes 2 and 5) or plus 10 μM GTPγS and 5 mM MgCl2 (lanes 3 and 6). Purified Gt was loaded as a control in lane 7. The bound samples were analyzed by SDS–PAGE followed by immunoblotting (IB) for Gβ using anti-Gβ antibody (top panel). A Coomassie blue-stained gel in the bottom panel shows that equal amounts of the GST and GST–Tctex-1 were used. (B) Gβγ and Tctex-1 co-IP in HEK cells. HEK cells were transfected with FLAG-tagged Gβ1 and Tctex-1 cDNAs as indicated and cell lysates were immunoprecipitated (IP) with anti-Tctex-1 antibody (top panel) or with anti-FLAG antibody (bottom panel) and subjected to SDS–PAGE, followed by Western blot analysis (IB) using anti-Gβ and anti-Tctex-1 antibodies. A 20 μg weight of total cell lysate was analyzed as input (right half of each panel) to show expression levels of Gβ and Tctex-1. The doublet seen in the anti-Gβ blots correspond to the endogenous Gβ and the FLAG-tagged Gβ proteins. (C) Tctex-1 interacts with several overexpressed Gβ isoforms, and with endogenous Gβ in brain lysates. HEK293 cells were cotransfected with FLAG-tagged Gβ1, β2, β3, β5 or β5L, along with Gγ1 or Gγ2 and Tctex-1 expression vectors. Tctex-1 was IP with anti-Tctex-1 antibody and the immunocomplexes were analyzed by Western blotting using anti-Tctex-1 antibody to detect Tctex-1 and anti-FLAG antibody to detect the FLAG-tagged Gβ subunits that co-IP with Tctex-1. Endogenous Tctex-1 was immunoprecipitated from E15 embryonic mouse brain lysate using anti-Tctex-1 antibody and analyzed by Western blotting for Tctex-1 and the associated brain Gβ subunit using anti-Tctex-1 and anti-pan Gβ antibody, respectively. A 20 μg weight of total cell lysate was analyzed by as input to show protein expression levels of Tctex-1, FLAG-tagged Gβ and endogenous Gβ using anti-Tctex-1, anti-FLAG and anti-pan Gβ antibodies, respectively. All images are representative of three independent experiments. Download figure Download PowerPoint To map the Gβγ-binding site on Tctex-1, we constructed N- and C-terminal truncation mutants of Tctex-1. FLAG-tagged full-length Tctex-1 and various truncation mutants of Tctex-1 were cotransfected with Gβ1 in HEK cells (Figure 2A). As shown in Figure 2B, full-length and the N-terminal truncated mutants of Tctex-1 were able to co-IP Gβγ, whereas the C-terminal truncation mutant of Tctex-1, 1–92, which is missing the last 21 amino acids of Tctex-1, failed to co-IP Gβγ (Figure 2B). Taken together, these results suggest that the C-terminal tail of Tctex-1 is required for the Gβγ–Tctex-1 interaction. Figure 2.Mapping of the Gβ-binding domain of Tctex-1. (A) Schematic representation of the N- and C-terminal truncation mutants of Tctex-1 used in the mapping of the Gβ-binding domain of Tctex-1. (B) C-terminal region of Tctex-1 is required for Gβ binding. HEK cells were cotransfected with the indicated truncation mutants of FLAG-tagged Tctex-1, along with Gβ cDNAs. Tctex-1 and its mutants were immunoprecipitated with anti-FLAG antibody and the immunocomplexes were analyzed by Western blotting with anti-Gβ antibody (top panels) and anti-FLAG antibody to detect FLAG-tagged Tctex-1 (bottom panels). The expression level of each protein was analyzed by direct Western using 20 μg of total cell lysate as input. This is a representative image of at least three independent experiments. Download figure Download PowerPoint Consensus binding motif on Gβ is required for Tctex-1 binding An 11-amino-acid peptide derived from DIC was used to identify residues on Tctex-1 that are involved in the DIC–Tctex-1 interaction (Mok et al, 2001). Comparison of this peptide sequence of DIC with sequences of other Tctex-1 target proteins helped to identify a motif of basic residues, R/K-R/K-X-X-R/K, as a consensus Tctex-1-binding motif (Mok et al, 2001). Additionally, a second Tctex-1-binding motif, V-S-K/H-T/S-X-V/T-T/S-N/Q-V, has also been identified in a subset of Tctex-1-interacting proteins (Sugai et al, 2003). We found a potential Tctex-1-binding motif in all six Gβ subunits, which maps to the outermost β-strand of the seventh blade of the Gβ propeller (Figure 3A and B). We generated a Gβ-peptide corresponding to amino acids 40–57 of Gβ1, which includes the basic Tctex-1-binding motif and surrounding residues either with no tag or with a thiol-specific, environmentally-sensitive, fluorescent compound, MIANS. We performed fluorescence anisotropy measurements to determine the binding affinity of the Gβ-peptide to Tctex-1. Keeping the MIANS-labeled Gβ-peptide constant and adding increasing concentrations of purified Tctex-1, we observed enhanced binding, which leveled off at high concentrations of Gβ-peptide (Figure 3C). The experimentally determined Kd for the Gβ-peptide–Tctex-1 interaction was 1.35 μM. Figure 3.Analysis of the Tctex-1-interacting motif on Gβ. (A) Structure of heterotrimer Gαβγ highlighting the Tctex-1-binding motif in Gβ. Gα and Gγ (gray) are shown as molecular surface representation, whereas Gβ (cyan) is shown as a secondary structure cartoon. Four β-strands that make up blade 7 of the Gβ propeller are highlighted (β-strands A–C in yellow and β-strand D in orange, left panel). Expanded region (right panel) shows the region of Gβ involved in the formation of blade 7. C-terminal residues 315–340 (yellow) form the A, B and C β-strands, whereas N-terminal residues 47–52 (orange) form the outermost D β-strand of this blade. Residues 47–52 comprise the consensus R/K-R/K-X-X-R/K Tctex-1-binding motif. Image created using VMD software (Humphrey et al, 1996). Crystal coordinates obtained from PDB file 1GOT (Lambright et al, 1996). (B) Consensus sequence R/K-R/K-X-X-R/K found in various Tctex-1-interacting proteins. (C) Fluorescence anisotropy of MIANS-labeled Gβ-peptide in the presence of Tctex-1. Tctex-1 was titrated to the MIANS Gβ-peptide, whose concentration was maintained at 0.7 μM. With increasing concentration of Tctex-1, there was an enhancement in the anisotropy value, indicative of specific binding of the peptide to the protein. In addition, the plot showed a sigmoidal curve, indicative of cooperative binding. Each data point is an average of five readings with standard error less than 5%. The Kd value for the Tctex-1-Gβ–peptide interaction from nonlinear regression analysis was calculated to be 1.35 μM. (D) Reduced interaction of Gβ AAA mutant with Tctex-1. HEK cells were cotransfected with Tctex-1 and the indicated FLAG-tagged Gβ mutants. The AAA Gβ1 mutant, wherein all three Arg residues (R48, R49 and R52) within the consensus Tctex-1-binding motif are mutated to alanine, showed significantly reduced ability to co-IP Tctex-1. The upper band observed in the top left panel of the anti-FLAG IP corresponds to the IgG heavy chain. This image is representative of three independent experiments. Download figure Download PowerPoint We further dissected the binding motif to identify which particular residues within the binding motif are critical for the Gβ–Tctex-1 interaction. The cluster of basic residues within the Tctex-1-binding motif (R/K-R/K-X-X-R/K) prompted us to evaluate the role of charge-based regulation of the interaction. We generated site-directed mutants of the Tctex-1-binding motif in Gβ, where the Arg residues were replaced either individually or in combination, and tested their ability to interact with Tctex-1 in co-IP experiments. In accordance with our hypothesis, we found that mutation of all three basic residues (R48, R49 and R52) to alanines within the full-length Gβ protein (Gβ1 AAA) significantly decreased the ability of Gβγ to interact with Tctex-1 (Figure 3D). Gβγ and Tctex-1 distribution overlaps in cultured hippocampal neurons Tctex-1 was recently shown to regulate neurite outgrowth in primary hippocampal neurons (Chuang et al, 2005). We decided to investigate whether the endogenous Gβγ–Tctex-1 complex, isolated from mouse brain lysates, could play a role in Tctex-1-induced neuritogenesis. To this end, we first examined the expression pattern of Gβ in cultured primary hippocampal neurons. Primary hippocampal neurons adopt the characteristic polarized morphology in a well-defined sequence of developmental stages (Dotti et al, 1988; Fukata et al, 2002). Within 24 h of plating, hippocampal neurons send out several processes of relatively equal length (stage 2). Even though phenotypically the neurons still appear non-polarized, proteins known to be involved in cell polarization already start to display a polarized distribution pattern (Bradke and Dotti, 2000; Fukata et al, 2002; Banker, 2003; Schwamborn and Puschel, 2004). At this stage, Gβ displays diffuse labeling throughout the cell body and the minor processes (Figure 4A–C). As the neurons develop through stages 2–3 and reach stage 3, they adopt the final differentiated polarized neuronal phenotype with one long neurite (the nascent axon) and several minor processes. Approximately 40% of stage 3 neurons examined (n>50) displayed Gβ immunoreactivity in the central region of the axonal growth cones (Figure 4D–K), whereas the remaining neurons failed to show an enhanced labeling intensity of Gβ in the growth cones (Figure 4L–N). Figure 4.Expression pattern of Gβ in various stages of cultured primary hippocampal neuron differentiation. Gβ distributes diffusely throughout the cell body and the minor processes in stage 2 neurons (A–C). As the neurons develop through stages 2–3 and reach stage 3 and adopt the well-differentiated neuronal phenotype, Gβ shows two distinct labeling patterns. Approximately 40% of stage 3 neurons (n>50) showed Gβ labeling in the central region of the axonal growth cone (D–K). An example of a stage 3 neuron that failed to show any detectable enhancement of Gβ in the growth cones is represented (L–N). Neurons were colabeled for Gβ (green in A, D, H and L), Tyr-Tubulin (red in B, E, I and M) and actin (blue in G and K). Overlayed images are shown in C, F, J and N. Scale bars equal 10 μm in panels (A–C) and 20 μm in panels (D–N). Download figure Download PowerPoint We then examined the expression patterns of Gβ and Tctex-1 in hippocampal neurons of various stages. Tctex-1 and Gβ are expressed essentially homogenously throughout the cell body and processes of stage 2 neurons (Figure 5Aa–c). As the neurons develop through stages 2–3, Gβ and Tctex-1 continue to overlap in the perinuclear Golgi region within the cell body. We also observed a strong colocalization of Tctex-1 and Gβ within the axonal growth cones of majority of stage 3 neurons (70%, n=100) (Figure 5Ad–i and 5B). Figure 5.Overlapping distribution of Tctex-1 and Gβ in hippocampal neurons. (A) Cultured hippocampal neurons in various stages of differentiation were colabeled for Gβ (green in a, d and g) and Tctex-1 (red in b, e and h). The overlayed images are shown in c, f and i. Gβ and Tctex-1 show homogenous expression within the cell body and all the neurites in typical stage 2 cells (a–c). As the neurons progress through stage 3, Gβ and Tctex-1 continue to overlap within the cell body, but in addition, show strong co-distribution at the growth cones of the future axon (a–i). Majority of the stage 3 neurons examined (70%, n=100) (a–f) show enhanced colabeling of Gβ and Tctex-1 in axonal growth cones as compared with the minor neurites that do not show an enrichment of Gβ or Tctex-1 at the tips. A small subset of the stage 3 neurons examined (30%, n=100) continued to show colabeling of Gβ and Tctex-1 in the cell body and at the axonal growth cones, and also showed some colabeling at the tips of the minor neurites (g–i). A magnified view of the growth cone of a stage 3 neuron is shown in the inset (g–i) Greater than 100 individual neurons were examined. Scale bar equals 10 μm (a–i) and 3 μm in the magnified insets (g–i). (B) Confocal image of a representative stage 3 neuron shows perinuclear, cytoplasmic staining for Gβ and Tctex-1 within the cell body and an enrichment at the tips of some of the axonal growth cones (panels A–C). Scale bar equals 10 μm. Download figure Download PowerPoint Role of Gβγ and Tctex-1 in neuronal differentiation To study the role of Gβγ in hippocampal neuronal differentiation, we used a Gβγ-specific antagonist, βARKct, to perform loss-of-function experiments. βARKct contains the Gβγ-binding domain of β-adrenergic receptor kinase 1 and when expressed in intact cells, it inhibits Gβγ-dependent signaling by binding to and sequestering Gβγ (Koch et al, 1994). Hippocampal neurons transfected with EGFP expression vector alone were indistinguishable from non-transfected cells and developed normally through stage 2 and stage 3 (Figure 6A). Quantification of the different stages showed that a significant fraction of neurons transfected with EGFP alone were found in stage 2 (52%) and stage 3 (40%) (Table I). To study the effect of βARKct on neurite outgrowth, hippocampal neurons were transfected either 2 h after plating or 12 h after plating with expression vectors for βARKct together with EGFP (Figure 6B and Supplementary Figure 1). As seen in Figure 6B, neurons that were transfected with the EGFP plus βARKct 2 h after plating failed to develop neurites and seemed to be arrested in stage 1 (78%) (Figure 6B and Supplementary Figure 1). The neighboring, untransfected cells (observed within the same field) appear healthy and proceed normally through development reaching stage 3 (Figure 6B). As compared with EGFP alone or un-transfected cells, a significantly smaller fraction of βARKct-transfected cells reached stage 2 (17%) or stage 3 (4%) (Table I). Neurons transfected 12 h after plating seemed to be arrested in stage 2 (Figure 6B). This result is very similar to what was seen with loss of Tctex-1 function wherein most Tctex-1-suppressed neurons had segmented lamellipodia but neither typical neurites nor growth cones (Chuang et al, 2005). Figure 6.Role of Gβγ and Tctex-1 in neuronal differentiation. (A) Ectopic expression of Gβ1 and Tctex-1 induces multiple, long neurites in hippocampal neurons. Hippocampal neurons were transfected as indicated, with expression vectors for GFP, GFP+FLAG-Gβ1, GFP+FLAG-Tctex-1 and GFP+Tctex-1+βARKct 2 h after plating. The transfected neurons were fixed and processed for GFP fluorescence 24 h after transfection. (B) Expression of Gβγ-sequestering reagent, βARKct inhibits neurite outgrowth. Hippocampal neurons were transfected either 2 h or 12 h after plating, with expression vectors for GFP and βARKct and analyzed 24 h after transfection for GFP expression (green) and Tyr-Tubulin labeling (red). βARKct expression results in arresting the cells in stage 1 when transfected 2 h after plating and in stage 2 when transfected 12 h after plating. Note that, under both conditions, the untransfected cells within the same field appear healthy and have reached stage 3. The images shown here are representative of three independent transfections. Quantification of the images from these transfections is shown in Table I. Download figure Download PowerPoint Table 1. Quantitative analyses of morphological changes of transfected neurons Overexpressed protein % cells (stage 1) % cells (stage 2) % cells with a single neurite (stage 3) % cells with multiple neurites >70–80 μm GFP alone 8±4 52±8 40±6 0.6±01 βARKct 78±6* 17±3* 4±2* ND FLAG-Gβ1 2±1 36±9* 42±14 22±6* FLAG-Tctex1 2±1 24±8* 28±6 38±8* FLAG-Tctex1+βARKct 10±4 38±4* 40±12 12±6* Cells were transfected at 2 h after plating and fixed 24 h later. Each transfection received 1 μg of GFP expressing vector for visualizing the transfected cells. For all other constructs, 2 μg of plasmid were typically used. The total amount of DNA added was kept constant by adding appropriate amount of control vector. A neurite longer than 70–80 μm was considered to be an axon in these analyses. Each value represents the mean±s.e.m. of at least 50–75 cells for each experimental condition. Asterisk represents value significantly different from that of the GFP-transfected group (P<0.01). ND, not detected. We then performed gain-of-function studies to examine the phenotype of neurons overexpressing Gβ1. Primary hippocampal neurons were transfected 2 h after plating with FLAG-Gβ1 and EGFP expression vectors. In contrast to cells expressing EGFP alone, a significantly greater number of FLAG-Gβ1-transfected" @default.
• W2022997685 created "2016-06-24" @default.
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• W2022997685 date "2007-05-10" @default.
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• W2022997685 title "G protein βγ subunit interaction with the dynein light-chain component Tctex-1 regulates neurite outgrowth" @default.
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