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Nerve growth factor induces neurite outgrowth of PC12 cells by promoting Gβγ-microtubule interaction.

Sierra-Fonseca JA, Najera O, Martinez-Jurado J, Walker EM, Varela-Ramirez A, Khan AM, Miranda M, Lamango NS, Roychowdhury S - BMC Neurosci (2014)

Bottom Line: We have found that NGF promoted the interaction of Gβγ with MTs and stimulated MT assembly.We found that these inhibitors disrupted Gβγ and ΜΤ organization and affected cellular morphology and neurite outgrowth.Altogether, our results demonstrate that βγ subunit of heterotrimeric G proteins play a critical role in neurite outgrowth and differentiation by interacting with MTs and modulating MT rearrangement.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Assembly and disassembly of microtubules (MTs) is critical for neurite outgrowth and differentiation. Evidence suggests that nerve growth factor (NGF) induces neurite outgrowth from PC12 cells by activating the receptor tyrosine kinase, TrkA. G protein-coupled receptors (GPCRs) as well as heterotrimeric G proteins are also involved in regulating neurite outgrowth. However, the possible connection between these pathways and how they might ultimately converge to regulate the assembly and organization of MTs during neurite outgrowth is not well understood.

Results: Here, we report that Gβγ, an important component of the GPCR pathway, is critical for NGF-induced neuronal differentiation of PC12 cells. We have found that NGF promoted the interaction of Gβγ with MTs and stimulated MT assembly. While Gβγ-sequestering peptide GRK2i inhibited neurite formation, disrupted MTs, and induced neurite damage, the Gβγ activator mSIRK stimulated neurite outgrowth, which indicates the involvement of Gβγ in this process. Because we have shown earlier that prenylation and subsequent methylation/demethylation of γ subunits are required for the Gβγ-MTs interaction in vitro, small-molecule inhibitors (L-28 and L-23) targeting prenylated methylated protein methyl esterase (PMPMEase) were tested in the current study. We found that these inhibitors disrupted Gβγ and ΜΤ organization and affected cellular morphology and neurite outgrowth. In further support of a role of Gβγ-MT interaction in neuronal differentiation, it was observed that overexpression of Gβγ in PC12 cells induced neurite outgrowth in the absence of added NGF. Moreover, overexpressed Gβγ exhibited a pattern of association with MTs similar to that observed in NGF-differentiated cells.

Conclusions: Altogether, our results demonstrate that βγ subunit of heterotrimeric G proteins play a critical role in neurite outgrowth and differentiation by interacting with MTs and modulating MT rearrangement.

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Related in: MedlinePlus

Overexpression of Gβγ induces neurite outgrowth in PC12 cells. PC12 cells were co-transfected with YFP-tagged constructs encoding (A) Gβ1 and Gγ2 (β1γ2) or with (B) Gβ1 and Gγ1 (β1γ1) in the absence of NGF, using Lipofectamine LTX PLUS reagent according to manufacturer instructions. Cells overexpressing fluorescent proteins were monitored at different time points (24, 48, and 72 h) for protein expression and morphological changes using a fluorescence microscope. Images taken with DIC and YFP filters are shown. (C) PC12 cells transfected with a plasmid-encoding YFP only was used as control and observed through the same time points. Neuronal processes, white arrows; growth cones, red arrows; axonal branching, broken white arrow; cytoskeletal labeling, white arrowhead; enlarged and bulky neurites, yellow arrows. (D and E) Neurites were traced and measured using the 2009 ZEN software from Zeiss. At least 100 cells from three independent experiments were measured for each preparation, and average neurite length and percent of cells bearing neurites calculations and statistical analysis were done using SigmaPlot software. (D) The average neurite length of Gβ1-, Gγ1-, Gγ2-, Gβ1γ1- and Gβ1γ2- overexpressing PC12 cells. (E) The percentage of cells bearing neurites in transfected cells was also estimated. *p value < 0.05; **p value < 0.005 when compared to control. #p value = 0.005 when compared with β1γ1.
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Fig6: Overexpression of Gβγ induces neurite outgrowth in PC12 cells. PC12 cells were co-transfected with YFP-tagged constructs encoding (A) Gβ1 and Gγ2 (β1γ2) or with (B) Gβ1 and Gγ1 (β1γ1) in the absence of NGF, using Lipofectamine LTX PLUS reagent according to manufacturer instructions. Cells overexpressing fluorescent proteins were monitored at different time points (24, 48, and 72 h) for protein expression and morphological changes using a fluorescence microscope. Images taken with DIC and YFP filters are shown. (C) PC12 cells transfected with a plasmid-encoding YFP only was used as control and observed through the same time points. Neuronal processes, white arrows; growth cones, red arrows; axonal branching, broken white arrow; cytoskeletal labeling, white arrowhead; enlarged and bulky neurites, yellow arrows. (D and E) Neurites were traced and measured using the 2009 ZEN software from Zeiss. At least 100 cells from three independent experiments were measured for each preparation, and average neurite length and percent of cells bearing neurites calculations and statistical analysis were done using SigmaPlot software. (D) The average neurite length of Gβ1-, Gγ1-, Gγ2-, Gβ1γ1- and Gβ1γ2- overexpressing PC12 cells. (E) The percentage of cells bearing neurites in transfected cells was also estimated. *p value < 0.05; **p value < 0.005 when compared to control. #p value = 0.005 when compared with β1γ1.

Mentions: To further elucidate the role of Gβγ in neuronal differentiation, we overexpressed Gβγ in PC12 cells. Since previous studies have indicated that Gβ1γ2 promoted MT assembly in vitro—and Gβ1γ1 was without any effect [24]—PC12 cells were transfected with either β1γ1 or β1γ2. YFP-tagged β1, γ2, or γ1 constructs were used for transfection. Cells were co-transfected with β1 and γ2, β1 and γ1, or individual constructs (Gβ1, Gγ1, and Gγ2). A plasmid encoding only YFP was used as control. Cells were monitored for protein expression and for possible neurite formation at different time points (24, 48, and 72 h). Both DIC and fluorescent images of the live cells are shown in Figure 6.Figure 6


Nerve growth factor induces neurite outgrowth of PC12 cells by promoting Gβγ-microtubule interaction.

Sierra-Fonseca JA, Najera O, Martinez-Jurado J, Walker EM, Varela-Ramirez A, Khan AM, Miranda M, Lamango NS, Roychowdhury S - BMC Neurosci (2014)

Overexpression of Gβγ induces neurite outgrowth in PC12 cells. PC12 cells were co-transfected with YFP-tagged constructs encoding (A) Gβ1 and Gγ2 (β1γ2) or with (B) Gβ1 and Gγ1 (β1γ1) in the absence of NGF, using Lipofectamine LTX PLUS reagent according to manufacturer instructions. Cells overexpressing fluorescent proteins were monitored at different time points (24, 48, and 72 h) for protein expression and morphological changes using a fluorescence microscope. Images taken with DIC and YFP filters are shown. (C) PC12 cells transfected with a plasmid-encoding YFP only was used as control and observed through the same time points. Neuronal processes, white arrows; growth cones, red arrows; axonal branching, broken white arrow; cytoskeletal labeling, white arrowhead; enlarged and bulky neurites, yellow arrows. (D and E) Neurites were traced and measured using the 2009 ZEN software from Zeiss. At least 100 cells from three independent experiments were measured for each preparation, and average neurite length and percent of cells bearing neurites calculations and statistical analysis were done using SigmaPlot software. (D) The average neurite length of Gβ1-, Gγ1-, Gγ2-, Gβ1γ1- and Gβ1γ2- overexpressing PC12 cells. (E) The percentage of cells bearing neurites in transfected cells was also estimated. *p value < 0.05; **p value < 0.005 when compared to control. #p value = 0.005 when compared with β1γ1.
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Related In: Results  -  Collection

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Fig6: Overexpression of Gβγ induces neurite outgrowth in PC12 cells. PC12 cells were co-transfected with YFP-tagged constructs encoding (A) Gβ1 and Gγ2 (β1γ2) or with (B) Gβ1 and Gγ1 (β1γ1) in the absence of NGF, using Lipofectamine LTX PLUS reagent according to manufacturer instructions. Cells overexpressing fluorescent proteins were monitored at different time points (24, 48, and 72 h) for protein expression and morphological changes using a fluorescence microscope. Images taken with DIC and YFP filters are shown. (C) PC12 cells transfected with a plasmid-encoding YFP only was used as control and observed through the same time points. Neuronal processes, white arrows; growth cones, red arrows; axonal branching, broken white arrow; cytoskeletal labeling, white arrowhead; enlarged and bulky neurites, yellow arrows. (D and E) Neurites were traced and measured using the 2009 ZEN software from Zeiss. At least 100 cells from three independent experiments were measured for each preparation, and average neurite length and percent of cells bearing neurites calculations and statistical analysis were done using SigmaPlot software. (D) The average neurite length of Gβ1-, Gγ1-, Gγ2-, Gβ1γ1- and Gβ1γ2- overexpressing PC12 cells. (E) The percentage of cells bearing neurites in transfected cells was also estimated. *p value < 0.05; **p value < 0.005 when compared to control. #p value = 0.005 when compared with β1γ1.
Mentions: To further elucidate the role of Gβγ in neuronal differentiation, we overexpressed Gβγ in PC12 cells. Since previous studies have indicated that Gβ1γ2 promoted MT assembly in vitro—and Gβ1γ1 was without any effect [24]—PC12 cells were transfected with either β1γ1 or β1γ2. YFP-tagged β1, γ2, or γ1 constructs were used for transfection. Cells were co-transfected with β1 and γ2, β1 and γ1, or individual constructs (Gβ1, Gγ1, and Gγ2). A plasmid encoding only YFP was used as control. Cells were monitored for protein expression and for possible neurite formation at different time points (24, 48, and 72 h). Both DIC and fluorescent images of the live cells are shown in Figure 6.Figure 6

Bottom Line: We have found that NGF promoted the interaction of Gβγ with MTs and stimulated MT assembly.We found that these inhibitors disrupted Gβγ and ΜΤ organization and affected cellular morphology and neurite outgrowth.Altogether, our results demonstrate that βγ subunit of heterotrimeric G proteins play a critical role in neurite outgrowth and differentiation by interacting with MTs and modulating MT rearrangement.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Assembly and disassembly of microtubules (MTs) is critical for neurite outgrowth and differentiation. Evidence suggests that nerve growth factor (NGF) induces neurite outgrowth from PC12 cells by activating the receptor tyrosine kinase, TrkA. G protein-coupled receptors (GPCRs) as well as heterotrimeric G proteins are also involved in regulating neurite outgrowth. However, the possible connection between these pathways and how they might ultimately converge to regulate the assembly and organization of MTs during neurite outgrowth is not well understood.

Results: Here, we report that Gβγ, an important component of the GPCR pathway, is critical for NGF-induced neuronal differentiation of PC12 cells. We have found that NGF promoted the interaction of Gβγ with MTs and stimulated MT assembly. While Gβγ-sequestering peptide GRK2i inhibited neurite formation, disrupted MTs, and induced neurite damage, the Gβγ activator mSIRK stimulated neurite outgrowth, which indicates the involvement of Gβγ in this process. Because we have shown earlier that prenylation and subsequent methylation/demethylation of γ subunits are required for the Gβγ-MTs interaction in vitro, small-molecule inhibitors (L-28 and L-23) targeting prenylated methylated protein methyl esterase (PMPMEase) were tested in the current study. We found that these inhibitors disrupted Gβγ and ΜΤ organization and affected cellular morphology and neurite outgrowth. In further support of a role of Gβγ-MT interaction in neuronal differentiation, it was observed that overexpression of Gβγ in PC12 cells induced neurite outgrowth in the absence of added NGF. Moreover, overexpressed Gβγ exhibited a pattern of association with MTs similar to that observed in NGF-differentiated cells.

Conclusions: Altogether, our results demonstrate that βγ subunit of heterotrimeric G proteins play a critical role in neurite outgrowth and differentiation by interacting with MTs and modulating MT rearrangement.

Show MeSH
Related in: MedlinePlus