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Proteins that promote filopodia stability, but not number, lead to more axonal-dendritic contacts.

Arstikaitis P, Gauthier-Campbell C, Huang K, El-Husseini A, Murphy TH - PLoS ONE (2011)

Bottom Line: We used a filopodia inducing motif that is found in GAP-43, as a molecular tool, and found this palmitoylated motif enhanced filopodia number and motility, but reduced the probability of forming a stable axon-dendrite contact.Conversely, expression of neuroligin-1 (NLG-1), a synapse inducing cell adhesion molecule, resulted in a decrease in filopodia motility, but an increase in the number of stable axonal contacts.Moreover, RNAi knockdown of NLG-1 reduced the number of presynaptic contacts formed.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry and The Brain Research Centre, University of British Columbia, Vancouver, British Columbia, Canada.

ABSTRACT
Dendritic filopodia are dynamic protrusions that are thought to play an active role in synaptogenesis and serve as precursors to spine synapses. However, this hypothesis is largely based on a temporal correlation between filopodia formation and synaptogenesis. We investigated the role of filopodia in synapse formation by contrasting the roles of molecules that affect filopodia elaboration and motility, versus those that impact synapse induction and maturation. We used a filopodia inducing motif that is found in GAP-43, as a molecular tool, and found this palmitoylated motif enhanced filopodia number and motility, but reduced the probability of forming a stable axon-dendrite contact. Conversely, expression of neuroligin-1 (NLG-1), a synapse inducing cell adhesion molecule, resulted in a decrease in filopodia motility, but an increase in the number of stable axonal contacts. Moreover, RNAi knockdown of NLG-1 reduced the number of presynaptic contacts formed. Postsynaptic scaffolding proteins such as Shank1b, a protein that induces the maturation of spine synapses, increased the rate at which filopodia transformed into spines by stabilization of the initial contact with axons. Taken together, these results suggest that increased filopodia stability and not density, may be the rate-limiting step for synapse formation.

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Specific synapse-inducing proteins are important for filopodia induction.(A) Schematic of the various fluorescently tagged constructs used in this study. SS-signal sequence, GFP-green fluorescent protein, HA- hemagglutinin (B) Representative images demonstrating filopodia induction by GAP 1–14, Cdc42 (CA)-Palm, NLG-1 and Shank1b. Neurons were transfected at DIV 6–7 and stained at DIV 8–9. (C) Quantification of the number of filopodia/100 µm shows that expression of GAP 1–14, Cdc42 (CA)-Palm and NLG-1 significantly increases filopodia number. In contrast, Shank1b failed to increase filopodia number. (D) Representative dendrites from neurons expressing GFP, GAP 1–14, Cdc42 (CA)-Palm, NLG-1 and Shank1b. (E) Quantification of the number of PSD-95 puncta expressed as a percentage that is normalized to control cells. Neurons expressing Cdc42 (CA)-Palm, NLG-1 and Shank1b showed an increase in the number of spines containing PSD95 puncta. In contrast, neurons expressing GAP 1–14 did not lead to an increase in the number of PSD-95 positive spines. (F) Quantification of PSD-95 puncta size. Neurons expressing NLG-1 and Shank1b showed an increase in the size of spines containing PSD95 puncta. In contrast, neurons expressing Cdc42 (CA)-Palm and GAP 1–14 showed no increase or a moderate increase in the size of PSD-95 puncta, respectively. 8–15 cells were analyzed for each group and were collected from 3 independent experiments. *p<0.05, **p<0.01, ***p<0.001. Data represent mean ±SEM. Scale bars, 10 µm.
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pone-0016998-g001: Specific synapse-inducing proteins are important for filopodia induction.(A) Schematic of the various fluorescently tagged constructs used in this study. SS-signal sequence, GFP-green fluorescent protein, HA- hemagglutinin (B) Representative images demonstrating filopodia induction by GAP 1–14, Cdc42 (CA)-Palm, NLG-1 and Shank1b. Neurons were transfected at DIV 6–7 and stained at DIV 8–9. (C) Quantification of the number of filopodia/100 µm shows that expression of GAP 1–14, Cdc42 (CA)-Palm and NLG-1 significantly increases filopodia number. In contrast, Shank1b failed to increase filopodia number. (D) Representative dendrites from neurons expressing GFP, GAP 1–14, Cdc42 (CA)-Palm, NLG-1 and Shank1b. (E) Quantification of the number of PSD-95 puncta expressed as a percentage that is normalized to control cells. Neurons expressing Cdc42 (CA)-Palm, NLG-1 and Shank1b showed an increase in the number of spines containing PSD95 puncta. In contrast, neurons expressing GAP 1–14 did not lead to an increase in the number of PSD-95 positive spines. (F) Quantification of PSD-95 puncta size. Neurons expressing NLG-1 and Shank1b showed an increase in the size of spines containing PSD95 puncta. In contrast, neurons expressing Cdc42 (CA)-Palm and GAP 1–14 showed no increase or a moderate increase in the size of PSD-95 puncta, respectively. 8–15 cells were analyzed for each group and were collected from 3 independent experiments. *p<0.05, **p<0.01, ***p<0.001. Data represent mean ±SEM. Scale bars, 10 µm.

Mentions: Since filopodia have been documented to play a role in synapse formation and the transformation to dendritic spines [2], [9], [11] we compared the ability of the palmitoylated proteins GAP 1–14, Cdc42 (CA)-Palm tagged with GFP as well as the scaffolding molecules, NLG-1 and Shank1b to induce the formation of filopodia (Figure 1A). Recently, we identified the brain-specific isoform Cdc42 (CA)-Palm, which plays an important role in the formation of dendritic spines [22]. We therefore decided to compare the differential effects of these molecules in the induction of dendritic filopodia.


Proteins that promote filopodia stability, but not number, lead to more axonal-dendritic contacts.

Arstikaitis P, Gauthier-Campbell C, Huang K, El-Husseini A, Murphy TH - PLoS ONE (2011)

Specific synapse-inducing proteins are important for filopodia induction.(A) Schematic of the various fluorescently tagged constructs used in this study. SS-signal sequence, GFP-green fluorescent protein, HA- hemagglutinin (B) Representative images demonstrating filopodia induction by GAP 1–14, Cdc42 (CA)-Palm, NLG-1 and Shank1b. Neurons were transfected at DIV 6–7 and stained at DIV 8–9. (C) Quantification of the number of filopodia/100 µm shows that expression of GAP 1–14, Cdc42 (CA)-Palm and NLG-1 significantly increases filopodia number. In contrast, Shank1b failed to increase filopodia number. (D) Representative dendrites from neurons expressing GFP, GAP 1–14, Cdc42 (CA)-Palm, NLG-1 and Shank1b. (E) Quantification of the number of PSD-95 puncta expressed as a percentage that is normalized to control cells. Neurons expressing Cdc42 (CA)-Palm, NLG-1 and Shank1b showed an increase in the number of spines containing PSD95 puncta. In contrast, neurons expressing GAP 1–14 did not lead to an increase in the number of PSD-95 positive spines. (F) Quantification of PSD-95 puncta size. Neurons expressing NLG-1 and Shank1b showed an increase in the size of spines containing PSD95 puncta. In contrast, neurons expressing Cdc42 (CA)-Palm and GAP 1–14 showed no increase or a moderate increase in the size of PSD-95 puncta, respectively. 8–15 cells were analyzed for each group and were collected from 3 independent experiments. *p<0.05, **p<0.01, ***p<0.001. Data represent mean ±SEM. Scale bars, 10 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3049770&req=5

pone-0016998-g001: Specific synapse-inducing proteins are important for filopodia induction.(A) Schematic of the various fluorescently tagged constructs used in this study. SS-signal sequence, GFP-green fluorescent protein, HA- hemagglutinin (B) Representative images demonstrating filopodia induction by GAP 1–14, Cdc42 (CA)-Palm, NLG-1 and Shank1b. Neurons were transfected at DIV 6–7 and stained at DIV 8–9. (C) Quantification of the number of filopodia/100 µm shows that expression of GAP 1–14, Cdc42 (CA)-Palm and NLG-1 significantly increases filopodia number. In contrast, Shank1b failed to increase filopodia number. (D) Representative dendrites from neurons expressing GFP, GAP 1–14, Cdc42 (CA)-Palm, NLG-1 and Shank1b. (E) Quantification of the number of PSD-95 puncta expressed as a percentage that is normalized to control cells. Neurons expressing Cdc42 (CA)-Palm, NLG-1 and Shank1b showed an increase in the number of spines containing PSD95 puncta. In contrast, neurons expressing GAP 1–14 did not lead to an increase in the number of PSD-95 positive spines. (F) Quantification of PSD-95 puncta size. Neurons expressing NLG-1 and Shank1b showed an increase in the size of spines containing PSD95 puncta. In contrast, neurons expressing Cdc42 (CA)-Palm and GAP 1–14 showed no increase or a moderate increase in the size of PSD-95 puncta, respectively. 8–15 cells were analyzed for each group and were collected from 3 independent experiments. *p<0.05, **p<0.01, ***p<0.001. Data represent mean ±SEM. Scale bars, 10 µm.
Mentions: Since filopodia have been documented to play a role in synapse formation and the transformation to dendritic spines [2], [9], [11] we compared the ability of the palmitoylated proteins GAP 1–14, Cdc42 (CA)-Palm tagged with GFP as well as the scaffolding molecules, NLG-1 and Shank1b to induce the formation of filopodia (Figure 1A). Recently, we identified the brain-specific isoform Cdc42 (CA)-Palm, which plays an important role in the formation of dendritic spines [22]. We therefore decided to compare the differential effects of these molecules in the induction of dendritic filopodia.

Bottom Line: We used a filopodia inducing motif that is found in GAP-43, as a molecular tool, and found this palmitoylated motif enhanced filopodia number and motility, but reduced the probability of forming a stable axon-dendrite contact.Conversely, expression of neuroligin-1 (NLG-1), a synapse inducing cell adhesion molecule, resulted in a decrease in filopodia motility, but an increase in the number of stable axonal contacts.Moreover, RNAi knockdown of NLG-1 reduced the number of presynaptic contacts formed.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry and The Brain Research Centre, University of British Columbia, Vancouver, British Columbia, Canada.

ABSTRACT
Dendritic filopodia are dynamic protrusions that are thought to play an active role in synaptogenesis and serve as precursors to spine synapses. However, this hypothesis is largely based on a temporal correlation between filopodia formation and synaptogenesis. We investigated the role of filopodia in synapse formation by contrasting the roles of molecules that affect filopodia elaboration and motility, versus those that impact synapse induction and maturation. We used a filopodia inducing motif that is found in GAP-43, as a molecular tool, and found this palmitoylated motif enhanced filopodia number and motility, but reduced the probability of forming a stable axon-dendrite contact. Conversely, expression of neuroligin-1 (NLG-1), a synapse inducing cell adhesion molecule, resulted in a decrease in filopodia motility, but an increase in the number of stable axonal contacts. Moreover, RNAi knockdown of NLG-1 reduced the number of presynaptic contacts formed. Postsynaptic scaffolding proteins such as Shank1b, a protein that induces the maturation of spine synapses, increased the rate at which filopodia transformed into spines by stabilization of the initial contact with axons. Taken together, these results suggest that increased filopodia stability and not density, may be the rate-limiting step for synapse formation.

Show MeSH
Related in: MedlinePlus