Development of the axonal βII-spectrin periodic skeleton requires active cytoskeletal remodelling
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eLife Assessment
This study examines how the neuronal cytoskeleton contributes to the formation of the axonal membrane-associated periodic skeleton (MPS) in embryonic dorsal root ganglia (DRG) neurons, using STED imaging. Conclusions are supported by convincing methods, data, and analyses. This useful work confirms previous data and improves our understanding of the roles of microtubules and actin dynamics in the chronological recruitment of MPS components.
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Abstract
Abstract
The axonal membrane-associated periodic skeleton (MPS), consisting of F-actin rings crosslinked by spectrin heterotetramers, is ubiquitous and critical for neuronal function and homoeostasis. However, the initiation and early development of the axonal MPS are poorly understood. Using superresolution imaging, we show that βII-spectrin is recruited early to the axonal cortex, followed by progressive establishment of long-range periodic order. Microtubule dynamics are essential for MPS formation in the early stages, but transition to a passive stabilising role in mature axons. We show that the early subplasmalemmal recruitment of βII-spectrin is dependent on cortical actin but not on actomyosin contractility, and active nucleation of F-actin is required in early development but is dispensable for the mature MPS. Using a βII-spectrin knockout model, we demonstrate that the actin-binding and lipid-interacting domains of βII-spectrin are critical for its subplasmalemmal confinement and, subsequently, MPS maturation. These findings highlight stage-specific cytoskeletal remodelling underlying MPS development and advance our understanding of axonal subcellular architecture.
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eLife Assessment
This study examines how the neuronal cytoskeleton contributes to the formation of the axonal membrane-associated periodic skeleton (MPS) in embryonic dorsal root ganglia (DRG) neurons, using STED imaging. Conclusions are supported by convincing methods, data, and analyses. This useful work confirms previous data and improves our understanding of the roles of microtubules and actin dynamics in the chronological recruitment of MPS components.
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Reviewer #1 (Public review):
The axonal membrane periodic skeleton (MPS) comprises axially aligned tetramers of α and β spectrins that are attached to evenly distributed radial F-actin rings, which maintain a
typical spacing of 180 - 190 nm. The exact molecular mechanisms underlying the early organization have been unclear. The focus of this study is on those mechanisms.This is a comprehensive and professionally carried out study. It brings convincing evidence that intact actin and microtubules are required for normal development of MPS and that the actin-binding and lipid-interacting domains of βII-spectrin are critical for its subplasmalemmal confinement and, subsequently, MPS maturation. However, whilst the study does bring new insights, we are still missing the overall understanding of how everything comes together.
The study …
Reviewer #1 (Public review):
The axonal membrane periodic skeleton (MPS) comprises axially aligned tetramers of α and β spectrins that are attached to evenly distributed radial F-actin rings, which maintain a
typical spacing of 180 - 190 nm. The exact molecular mechanisms underlying the early organization have been unclear. The focus of this study is on those mechanisms.This is a comprehensive and professionally carried out study. It brings convincing evidence that intact actin and microtubules are required for normal development of MPS and that the actin-binding and lipid-interacting domains of βII-spectrin are critical for its subplasmalemmal confinement and, subsequently, MPS maturation. However, whilst the study does bring new insights, we are still missing the overall understanding of how everything comes together.
The study describes, using spectrin mutations, that the membrane and actin binding of spectrin are required for the proper organization of MPS. However, it is unclear how everything could come together mechanistically.
The authors follow how the MPS is organized by looking at spectrin. Latrunculin affects actin polymerization, as well as CK666 and formin inhibition, but it remains unclear which actin structures are affected. The same is true for microtubules; while they are affected, we don't know how they are affected.
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Reviewer #2 (Public review):
Summary:
In their manuscript, Bodas et al present a chronological analysis of the development of the axonal MPS in embryonic DRG neurons, using a series of biochemical assays coupled with STED nanoscopy. Several interesting conclusions, well supported by the data presented, are drawn that further our understanding of bII-spectrin axonal recruitment and on the role of microtubules and actin dynamics during the early MPS formation and at the latter stages of neuronal maturation.
Strengths:
The assays presented are well-designed, and the results obtained clearly support the main conclusions drawn by the authors. Their findings highlight important aspects of cytoskeleton regulation and dynamics required for MPS formation/maintenance, i.e, during different stages of neuronal development, that remained undocumented.
Reviewer #2 (Public review):
Summary:
In their manuscript, Bodas et al present a chronological analysis of the development of the axonal MPS in embryonic DRG neurons, using a series of biochemical assays coupled with STED nanoscopy. Several interesting conclusions, well supported by the data presented, are drawn that further our understanding of bII-spectrin axonal recruitment and on the role of microtubules and actin dynamics during the early MPS formation and at the latter stages of neuronal maturation.
Strengths:
The assays presented are well-designed, and the results obtained clearly support the main conclusions drawn by the authors. Their findings highlight important aspects of cytoskeleton regulation and dynamics required for MPS formation/maintenance, i.e, during different stages of neuronal development, that remained undocumented.
Weaknesses:
The study is mostly limited to biochemical assays followed by STED microscopy to analyse MPS periodicity and (in certain cases) axonal diameter. Functional implications of the manipulations done are lacking, as well as analyses of axonal integrity/degeneration. This is a relevant aspect, as some of the effects observed may be a secondary effect of decreased neuronal/axonal viability.
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Reviewer #3 (Public review):
Summary:
In this study, Shivani Bodas et al. investigate the role of actin, actin-binding proteins, and microtubules in regulating the membrane-associated periodic skeleton (MPS) in neuronal axons. The MPS, first reported by Ke Xu et al. in 2013 (Science), has since been implicated in various neuronal functions, including mechanical support, axonal diameter control, axonal degeneration regulation, and spatial organization of signaling molecules. Given its biological importance, further elucidation of MPS assembly mechanisms is of considerable interest. However, I have concerns regarding the novelty and strength of the conclusions presented in this work. Many of the findings largely reiterate previously published observations, and the most novel conclusions are not fully substantiated by the data.
Strengths:
(…
Reviewer #3 (Public review):
Summary:
In this study, Shivani Bodas et al. investigate the role of actin, actin-binding proteins, and microtubules in regulating the membrane-associated periodic skeleton (MPS) in neuronal axons. The MPS, first reported by Ke Xu et al. in 2013 (Science), has since been implicated in various neuronal functions, including mechanical support, axonal diameter control, axonal degeneration regulation, and spatial organization of signaling molecules. Given its biological importance, further elucidation of MPS assembly mechanisms is of considerable interest. However, I have concerns regarding the novelty and strength of the conclusions presented in this work. Many of the findings largely reiterate previously published observations, and the most novel conclusions are not fully substantiated by the data.
Strengths:
(1) The MPS represents a structurally and functionally important cytoskeletal system in neurons. Studies aimed at understanding its developmental mechanisms are biologically meaningful and potentially impactful.
(2) The authors attempt to dissect MPS assembly during early neuronal development, a process that could offer mechanistic insight into how the MPS is established and maintained.
Weaknesses:
(1) Limited Novelty Across Results Sections:
Of the seven Results sections, only one (Figure 6) and part of another (Figure 9) present data leading to relatively novel interpretations, specifically, the authors' claim that βII-spectrin is recruited to the axonal cortex via F-actin interactions as early as DIV1, followed by rearrangement into a periodic structure by DIV4. However, this conclusion is not fully supported (see below). The remaining results (Figures 1-5, 7, and 8) largely recapitulate findings reported in earlier studies and thus add limited new knowledge.
(2) Insufficient Evidence for Early Recruitment and Rearrangement of βII-spectrin:
The claim that βII-spectrin is recruited to the axonal cortex via F-actin interactions as early as at DIV 1 and subsequently reorganized into a periodic structure during DIV1-4 is central to the manuscript but lacks robust experimental support.
On Page 17, Line 526, the authors the authors state that " To exclude cytoplasmic spectrin resulting from overexpression, only axons with low expression of βII spectrin-GFP were selected for the analysis". However, selecting for low expression alone does not guarantee the absence of cytoplasmic signal. Without volumetric imaging (e.g., 3D super-resolution imaging to see the cross section of axons), it is difficult to definitively conclude that the FRAP data (Figures 6 and 9) reflect cortical rather than cytoplasmic localization.
Prior FRAP studies (Zhong et al., eLife 2014) observed minimal fluorescence recovery over 1800 seconds in axons expressing βII-spectrin-GFP at low levels, with faster recovery (~200-300 seconds) only evident under high expression conditions. The fast recovery kinetics (tens of seconds) reported in this manuscript could plausibly result from free diffusion of cytoplasmic βII-spectrin-GFP rather than cortical turnover.
Furthermore, on Page 10, Line 310, the authors assert that endogenous βII-spectrin "is recruited early to the axonal cortex, followed by progressive establishment of periodic order". However, the STED images shown in Figure 1 do not convincingly distinguish between cortical and cytoplasmic pools.
As such, the observed disordered βII-spectrin molecules, whether overexpressed or endogenous, could still represent a diffuse cytoplasmic population. An alternative and perhaps more parsimonious interpretation is that βII-spectrin is initially cytoplasmic and only later recruited and arranged into periodic structures at the cortex.
(3) Use of Pharmacological Perturbations:
Like many earlier studies, this manuscript relies heavily on pharmacological perturbation (e.g., cytoskeletal drugs) to assess the roles of actin, actin-binding proteins, and microtubules in MPS assembly. While this approach is widely used, it is important to acknowledge that such agents may have off-target effects. The manuscript would benefit from greater caution in interpreting these results, or better yet, the inclusion of genetic or optogenetic approaches to independently validate these findings.
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