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Localized regulation of the axon shaft during the emergence of collateral branches.

Gallo G - Neural Regen Res (2015)

View Article: PubMed Central - PubMed

Affiliation: Shriners Hospitals Pediatric Research Center, Temple University School of Medicine, Department of Anatomy and Cell Biology, Philadelphia, PA, USA.

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Following NGF treatment, sites of patch formation correlate with the presence of the high affinity TrkA receptor for NGF (Ketschek and Gallo, 2010)... However, TrkA receptors are found in clusters all along the axon and not all clusters give rise to patches, indicating that mere receptor localization is not sufficient to determine where patches form... Importantly, the effects of NGF on microtubule splaying occurred maximally within 10 min of treatment and preceded the increase in the formation of actin patches and eventually branches... Thus, the splaying of axonal microtubules is one of the earliest features of the response of sensory axons as they begin to form branches following treatment with NGF... MAP1B was considered because prior studies determined that it is a negative regulator of axon branching... Analysis of the levels of microtubule associated MAP1B did not reveal any changes at sites of splaying relative to adjacent sites of the axon that did not exhibit splaying... In contrast, in the absence of NGF, MAP1B association with microtubules was high during all stages of branching... Thus, a yet to be fully elucidated mechanism downstream of NGF signaling decreases the association of MAP1B with microtubules during the early stages of branching... Insights into the possible mechanism regulating the association of MAP1B with microtubules during the early stages of branching came from investigation of the phosphorylated form of MAP1B at a glycogen synthase kinase 3β (GSK3β) site (threonine 1265)... Prior studies by other laboratories reported effects of NGF on GSK3β activity that are at face value in conflict... In our study, we used an antibody to MAP1B phosphorylated at the GSK3β site and analyzed its distribution and levels in axons through quantitative immunocytochemistry... Consistent with studies observing NGF-induced increases in MAP1B phosphorylation, we found that NGF treatment increased the net levels of phosphorylated MAP1B within the axon shaft by extending the coverage of the axon shaft exhibiting phosphorylated MAP1B... As phosphorylation of MAP1B by GSK3β is promotes microtubule tip polymerization and dynamics, these data indicate that the decreased phosphorylation of MAP1B in axonal filopodia may reflect a mechanism that contributes to the stabilization of microtubules during the early stages of branch formation... Our prior work has determined that the actin-based component of NGF-induced axon branching is dependent on NGF-induced intra-axonal protein synthesis (Spillane et al., 2012, 2013)... However, to date we have not yet found any effect of NGF on axonal microtubules which is dependent on intra-axonal proteins synthesis (Spillane et al., 2012).

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Overview of the mechanism of sensory axon collateral branching.(A) Sequence of cytoskeletal events underlying axon branching. The first step (1) is the formation of an actin patch, which serves as a precursor to the formation of an axonal filopodium (2; actin filaments shown in red). Next, the plus tip of an axonal microtubule (MT, green) must invade the filopodium and become stabilized (3). Finally, the filopodium undergoes maturation into a branch (4). During maturation the filopodium changes its morphology and actin distribution. The actin filament bundle that characterizes a filopodium is reorganized and the actin filaments become polarized to the tip of the nascent branch, which develops a small growth cone, and the shaft of the filopodium now contains microtubules. For actual examples of actin filament and microtubule distributions at the various stages of branch formation see Figure 2. (B) Time lapse sequence of actin patch formation and filopodia emergence as revealed by eYFP-β-actin imaging along embryonic sensory axons. Time in seconds is shown in panels. Bar = 1 μm. Between 3–15 seconds the actin patch (yellow arrowhead) forms and elaborates, i.e., grows in size and intensity. Between 15–21 seconds a filopodium emerges from the patch (red arrowhead). (C) Proposed conceptual stochastic model for the determination of the site of axon branching. Multiple basic cellular events have to occur in a correct spatio-temporal sequence in order for a branch to form. In the interest of simplicity, the schematic represents a subset of the fundamental required events in branch formation; the formation of axonal filopodia, the entry and stabilization of microtubules into axonal filopodia, and the presence of a stalled mitochondrion. Each one of these basic events is in turn dependent on a complex multi-step biochemical mechanism. In the context of the Ketschek et al (2015) work, the regulation of the phosphorylation of MAP1B by GSK3β in axonal filopodia may contribute to the stabilization of microtubules in filopodia. In the panel, axon segment number 1 “wins” all but the stabilization of the microtubules and fails to generate a branch. In contrast, axon segment number 2 “wins” all and is able to generate a branch. Image of slot machine used as royalty free stock image from www.dreamstime.com. eYFP: Enhanced yellow fluorescent protein; MAP1B: microtubule associated protein 1B; GSK3beta: glycogen synthase kinase 3beta.
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Figure 1: Overview of the mechanism of sensory axon collateral branching.(A) Sequence of cytoskeletal events underlying axon branching. The first step (1) is the formation of an actin patch, which serves as a precursor to the formation of an axonal filopodium (2; actin filaments shown in red). Next, the plus tip of an axonal microtubule (MT, green) must invade the filopodium and become stabilized (3). Finally, the filopodium undergoes maturation into a branch (4). During maturation the filopodium changes its morphology and actin distribution. The actin filament bundle that characterizes a filopodium is reorganized and the actin filaments become polarized to the tip of the nascent branch, which develops a small growth cone, and the shaft of the filopodium now contains microtubules. For actual examples of actin filament and microtubule distributions at the various stages of branch formation see Figure 2. (B) Time lapse sequence of actin patch formation and filopodia emergence as revealed by eYFP-β-actin imaging along embryonic sensory axons. Time in seconds is shown in panels. Bar = 1 μm. Between 3–15 seconds the actin patch (yellow arrowhead) forms and elaborates, i.e., grows in size and intensity. Between 15–21 seconds a filopodium emerges from the patch (red arrowhead). (C) Proposed conceptual stochastic model for the determination of the site of axon branching. Multiple basic cellular events have to occur in a correct spatio-temporal sequence in order for a branch to form. In the interest of simplicity, the schematic represents a subset of the fundamental required events in branch formation; the formation of axonal filopodia, the entry and stabilization of microtubules into axonal filopodia, and the presence of a stalled mitochondrion. Each one of these basic events is in turn dependent on a complex multi-step biochemical mechanism. In the context of the Ketschek et al (2015) work, the regulation of the phosphorylation of MAP1B by GSK3β in axonal filopodia may contribute to the stabilization of microtubules in filopodia. In the panel, axon segment number 1 “wins” all but the stabilization of the microtubules and fails to generate a branch. In contrast, axon segment number 2 “wins” all and is able to generate a branch. Image of slot machine used as royalty free stock image from www.dreamstime.com. eYFP: Enhanced yellow fluorescent protein; MAP1B: microtubule associated protein 1B; GSK3beta: glycogen synthase kinase 3beta.

Mentions: The formation of an axon branch is strictly dependent on both actin filaments and microtubules (Figures 1A and 2; see Kalil and Dent, 2014 for an excellent review). The first step in the formation of a branch, in vivo and in vitro, is the emergence of an axonal filopodium. Filopodia are finger-like projections from the surface of cells and are supported by a bundle of polymerizing actin filaments. The regulation of filopodia formation requires multiple regulatory mechanisms (Gallo, 2013), including Rho-family GTPases which are major regulators of the actin cytoskeleton (Spillane and Gallo, 2014). However, the majority of filopodia are transient and retracted back into the axon shaft without giving rise to a branch. For a filopodium to mature into a branch it must be invaded by microtubules, which provide structural stability and also allow for the transport of organelles and proteins into the nascent branch. However, as microtubule tips are dynamic, their entry into a filopodium is insufficient to promote the maturation of the filopodium into a branch and it is generally considered that a subsequent step involving the stabilization of the microtubule within the axonal filopodium is required for branch formation. Thus, in order to understand how a branch forms, and why it forms at a specific site, it is necessary to understand how the neuron locally regulates the dynamics and organization of actin filaments and microtubules.


Localized regulation of the axon shaft during the emergence of collateral branches.

Gallo G - Neural Regen Res (2015)

Overview of the mechanism of sensory axon collateral branching.(A) Sequence of cytoskeletal events underlying axon branching. The first step (1) is the formation of an actin patch, which serves as a precursor to the formation of an axonal filopodium (2; actin filaments shown in red). Next, the plus tip of an axonal microtubule (MT, green) must invade the filopodium and become stabilized (3). Finally, the filopodium undergoes maturation into a branch (4). During maturation the filopodium changes its morphology and actin distribution. The actin filament bundle that characterizes a filopodium is reorganized and the actin filaments become polarized to the tip of the nascent branch, which develops a small growth cone, and the shaft of the filopodium now contains microtubules. For actual examples of actin filament and microtubule distributions at the various stages of branch formation see Figure 2. (B) Time lapse sequence of actin patch formation and filopodia emergence as revealed by eYFP-β-actin imaging along embryonic sensory axons. Time in seconds is shown in panels. Bar = 1 μm. Between 3–15 seconds the actin patch (yellow arrowhead) forms and elaborates, i.e., grows in size and intensity. Between 15–21 seconds a filopodium emerges from the patch (red arrowhead). (C) Proposed conceptual stochastic model for the determination of the site of axon branching. Multiple basic cellular events have to occur in a correct spatio-temporal sequence in order for a branch to form. In the interest of simplicity, the schematic represents a subset of the fundamental required events in branch formation; the formation of axonal filopodia, the entry and stabilization of microtubules into axonal filopodia, and the presence of a stalled mitochondrion. Each one of these basic events is in turn dependent on a complex multi-step biochemical mechanism. In the context of the Ketschek et al (2015) work, the regulation of the phosphorylation of MAP1B by GSK3β in axonal filopodia may contribute to the stabilization of microtubules in filopodia. In the panel, axon segment number 1 “wins” all but the stabilization of the microtubules and fails to generate a branch. In contrast, axon segment number 2 “wins” all and is able to generate a branch. Image of slot machine used as royalty free stock image from www.dreamstime.com. eYFP: Enhanced yellow fluorescent protein; MAP1B: microtubule associated protein 1B; GSK3beta: glycogen synthase kinase 3beta.
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Figure 1: Overview of the mechanism of sensory axon collateral branching.(A) Sequence of cytoskeletal events underlying axon branching. The first step (1) is the formation of an actin patch, which serves as a precursor to the formation of an axonal filopodium (2; actin filaments shown in red). Next, the plus tip of an axonal microtubule (MT, green) must invade the filopodium and become stabilized (3). Finally, the filopodium undergoes maturation into a branch (4). During maturation the filopodium changes its morphology and actin distribution. The actin filament bundle that characterizes a filopodium is reorganized and the actin filaments become polarized to the tip of the nascent branch, which develops a small growth cone, and the shaft of the filopodium now contains microtubules. For actual examples of actin filament and microtubule distributions at the various stages of branch formation see Figure 2. (B) Time lapse sequence of actin patch formation and filopodia emergence as revealed by eYFP-β-actin imaging along embryonic sensory axons. Time in seconds is shown in panels. Bar = 1 μm. Between 3–15 seconds the actin patch (yellow arrowhead) forms and elaborates, i.e., grows in size and intensity. Between 15–21 seconds a filopodium emerges from the patch (red arrowhead). (C) Proposed conceptual stochastic model for the determination of the site of axon branching. Multiple basic cellular events have to occur in a correct spatio-temporal sequence in order for a branch to form. In the interest of simplicity, the schematic represents a subset of the fundamental required events in branch formation; the formation of axonal filopodia, the entry and stabilization of microtubules into axonal filopodia, and the presence of a stalled mitochondrion. Each one of these basic events is in turn dependent on a complex multi-step biochemical mechanism. In the context of the Ketschek et al (2015) work, the regulation of the phosphorylation of MAP1B by GSK3β in axonal filopodia may contribute to the stabilization of microtubules in filopodia. In the panel, axon segment number 1 “wins” all but the stabilization of the microtubules and fails to generate a branch. In contrast, axon segment number 2 “wins” all and is able to generate a branch. Image of slot machine used as royalty free stock image from www.dreamstime.com. eYFP: Enhanced yellow fluorescent protein; MAP1B: microtubule associated protein 1B; GSK3beta: glycogen synthase kinase 3beta.
Mentions: The formation of an axon branch is strictly dependent on both actin filaments and microtubules (Figures 1A and 2; see Kalil and Dent, 2014 for an excellent review). The first step in the formation of a branch, in vivo and in vitro, is the emergence of an axonal filopodium. Filopodia are finger-like projections from the surface of cells and are supported by a bundle of polymerizing actin filaments. The regulation of filopodia formation requires multiple regulatory mechanisms (Gallo, 2013), including Rho-family GTPases which are major regulators of the actin cytoskeleton (Spillane and Gallo, 2014). However, the majority of filopodia are transient and retracted back into the axon shaft without giving rise to a branch. For a filopodium to mature into a branch it must be invaded by microtubules, which provide structural stability and also allow for the transport of organelles and proteins into the nascent branch. However, as microtubule tips are dynamic, their entry into a filopodium is insufficient to promote the maturation of the filopodium into a branch and it is generally considered that a subsequent step involving the stabilization of the microtubule within the axonal filopodium is required for branch formation. Thus, in order to understand how a branch forms, and why it forms at a specific site, it is necessary to understand how the neuron locally regulates the dynamics and organization of actin filaments and microtubules.

View Article: PubMed Central - PubMed

Affiliation: Shriners Hospitals Pediatric Research Center, Temple University School of Medicine, Department of Anatomy and Cell Biology, Philadelphia, PA, USA.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Following NGF treatment, sites of patch formation correlate with the presence of the high affinity TrkA receptor for NGF (Ketschek and Gallo, 2010)... However, TrkA receptors are found in clusters all along the axon and not all clusters give rise to patches, indicating that mere receptor localization is not sufficient to determine where patches form... Importantly, the effects of NGF on microtubule splaying occurred maximally within 10 min of treatment and preceded the increase in the formation of actin patches and eventually branches... Thus, the splaying of axonal microtubules is one of the earliest features of the response of sensory axons as they begin to form branches following treatment with NGF... MAP1B was considered because prior studies determined that it is a negative regulator of axon branching... Analysis of the levels of microtubule associated MAP1B did not reveal any changes at sites of splaying relative to adjacent sites of the axon that did not exhibit splaying... In contrast, in the absence of NGF, MAP1B association with microtubules was high during all stages of branching... Thus, a yet to be fully elucidated mechanism downstream of NGF signaling decreases the association of MAP1B with microtubules during the early stages of branching... Insights into the possible mechanism regulating the association of MAP1B with microtubules during the early stages of branching came from investigation of the phosphorylated form of MAP1B at a glycogen synthase kinase 3β (GSK3β) site (threonine 1265)... Prior studies by other laboratories reported effects of NGF on GSK3β activity that are at face value in conflict... In our study, we used an antibody to MAP1B phosphorylated at the GSK3β site and analyzed its distribution and levels in axons through quantitative immunocytochemistry... Consistent with studies observing NGF-induced increases in MAP1B phosphorylation, we found that NGF treatment increased the net levels of phosphorylated MAP1B within the axon shaft by extending the coverage of the axon shaft exhibiting phosphorylated MAP1B... As phosphorylation of MAP1B by GSK3β is promotes microtubule tip polymerization and dynamics, these data indicate that the decreased phosphorylation of MAP1B in axonal filopodia may reflect a mechanism that contributes to the stabilization of microtubules during the early stages of branch formation... Our prior work has determined that the actin-based component of NGF-induced axon branching is dependent on NGF-induced intra-axonal protein synthesis (Spillane et al., 2012, 2013)... However, to date we have not yet found any effect of NGF on axonal microtubules which is dependent on intra-axonal proteins synthesis (Spillane et al., 2012).

No MeSH data available.