Recombinant Mouse Atlastin-1 (Atl1)

What is Mouse Atlastin-1 (Atl1) and what is its primary function?

Mouse Atlastin-1 (Atl1) is a GTPase belonging to the dynamin superfamily that plays a critical role in endoplasmic reticulum (ER) morphology. Functionally, Atl1 utilizes energy from GTP hydrolysis to facilitate membrane tethering and fusion, which promotes the formation of highly branched, smooth ER networks . This protein is particularly important for maintaining the three-way junctions that characterize normal ER morphology . Deficiency in Atlastin-1 results in altered ER morphology with reduced numbers of three-way junctions, demonstrating its essential role in ER structural maintenance .

The mouse Atl1 protein has several synonyms in scientific literature, including 4930435M24Rik, Adfsp, Fsp1, Spg3, and Spg3a . Like its human counterpart, mouse Atl1 is predominantly expressed in neuronal tissues and plays a crucial role in axonal maintenance.

How does Atlastin-1 expression vary across tissues and developmental stages?

Based on studies of human atlastin expression patterns, which share similarities with mouse models, there is a significant tissue-specific distribution of atlastin isoforms. Atlastin-1 is highly expressed in brain tissue but shows lower expression in non-neurological tissues . In contrast, atlastins-2 and -3 show minimal expression in brain tissue but are highly expressed in many non-neurological tissues .

What are the structural characteristics of Atlastin-1 that define its function?

Atlastin-1 has several key structural features that define its functional capabilities:

• GTPase Domain: The G domain is responsible for GTP binding and hydrolysis, which drives the conformational changes necessary for membrane fusion .

• Middle Domain: Works in conjunction with the G domain in the pre-hydrolysis state .

• Hypervariable Region (HVR): A distinct, isoform-specific structural feature that contributes to membrane tethering efficiency . Crystal structures reveal that the HVR of ATL1 makes direct contacts with the G domain of adjacent protomers, potentially coordinating the catalytic cycles of multiple atlastins .

• Transmembrane Domains: These anchor the protein to the ER membrane, with the majority of the protein (including the GTPase domain) facing the cytosol .

The protein can form homotetramers, which is important for its biological function . Crystal structures of ATL1 and ATL3 have revealed that these proteins adopt specific conformations related to their GTP binding and hydrolysis states, with the GDP-bound catalytic core of human ATL1 resolved at 2.2Å resolution .

How do mutations in Atlastin-1 contribute to neurological disorders?

Mutations in the ATL1 gene encoding Atlastin-1 are one of the most common causes of hereditary spastic paraplegia (HSP), a group of genetic neurodegenerative conditions characterized by distal axonal degeneration of the corticospinal tract axons . The disease primarily manifests as a predominantly childhood-onset HSP that may be pure but is often accompanied by peripheral neuropathy .

The mutational spectrum of atlastin-1-HSP is diverse, encompassing:

• Missense mutations: These tend to cluster at various points within key domains .

• Loss of function mutations: Including frameshift, nonsense, and whole exon deletion mutations .

• Novel mutations: Such as an in-frame insertion mutation leading to the addition of an asparagine residue at position 417 (N417ins), which is associated with complex, early-onset spastic quadriplegia affecting all four extremities, generalized dystonia, and thinning of the corpus callosum .

Most atlastin-1 mutations are thought to act via a dominant negative mechanism, although gain-of-function mutations have been associated with more severe phenotypes . The presence of loss-of-function mutations suggests that in some cases, a haploinsufficiency mechanism may operate .

What experimental models are most effective for studying Atlastin-1 function?

Several experimental models have proven valuable for investigating Atlastin-1 function:

• CRISPR-inhibition (CRISPRi) in human cortical neurons: This approach has been successfully used to generate human cortical neurons lacking atlastin-1 . The system involves using a neurogenic transcription factor gene (NGN2) under a doxycycline-responsive promoter, knocked into the AAVS1 safe harbor locus in the WTC11 stem cell line . This allows rapid, scalable, and reproducible differentiation to predominantly glutamatergic cortical neurons upon culture with doxycycline .

• Recombinant protein expression systems: Purified recombinant mouse Atl1 with C-terminal MYC/DDK tags can be expressed in HEK293T cells . The resulting protein has a molecular weight of 63.8 kDa and can be purified to >80% as determined by SDS-PAGE and Coomassie blue staining .

• Crystal structure analysis: Using selenomethionine-derivatized protein crystals grown from the GDP-bound catalytic core of human ATL1, researchers have collected data at resolutions of 2.2Å and 3.5Å, enabling detailed structural analysis .

• Low efficiency transfection with fluorescent proteins: This technique allows visualization of individual neuron morphology by transfecting neurons with vectors expressing eGFP and emerald fluorescent proteins before fixation and microscopy analysis .

What methodology should be used to assess Atlastin-1's role in ER morphology?

To assess Atlastin-1's role in ER morphology, researchers should consider the following methodological approach:

How can the GTPase activity of recombinant Atlastin-1 be measured and manipulated?

The GTPase activity of recombinant Atlastin-1 can be measured and manipulated using several approaches:

What is the relationship between Atlastin-1 and lysosomal function?

Recent research has revealed an unexpected relationship between Atlastin-1, traditionally viewed as an ER-shaping protein, and lysosomal function:

• Lysosomal proteolytic capacity: Neurons lacking atlastin-1 show reduced lysosomal proteolytic capacity . This suggests that proper ER morphology maintained by atlastin-1 is necessary for normal lysosomal function.

• Endosomal tubulation: Atlastin-1 deficient neurons exhibit longer endosomal tubules, indicating defective tubule fission . This altered endosomal morphology may contribute to the observed lysosomal dysfunction.

• Pathogenic mechanism in HSP: These findings strengthen the idea that defective lysosome function contributes to the pathogenesis of a broad group of hereditary spastic paraplegias, including those where the primary localization of the involved protein is not at the endolysosomal system .

This relationship highlights an important connection between ER morphology and endolysosomal function in neurons, suggesting that therapeutic approaches targeting lysosomal function might be beneficial in atlastin-1-related HSP.

How does the hypervariable region (HVR) contribute to Atlastin-1 function?

The hypervariable region (HVR) of Atlastin-1 has emerged as a crucial structural element with several important functions:

• Membrane tethering: The HVR of ATL1 contributes positively to membrane tethering efficiency . It appears to enhance the ability of the protein to bring ER membranes into close proximity for subsequent fusion.

• Oligomerization: Crystal structures show that the HVR of ATL1 makes direct contacts with the G domain of adjacent protomers . This interaction could facilitate the formation of HVR-dependent oligomers on a single membrane, potentially coordinating the catalytic cycles of multiple ATL proteins and priming them for interactions with other membranes .

• Post-translational regulation: The HVR is subject to post-translational regulation through phosphorylation-dependent modification . A kinase screen has identified candidates that specifically modify the HVR site, corresponding to modifications detected on ATL1 in cells .

• Isoform specificity: The HVR is a distinct, isoform-specific structural feature that likely contributes to the specialized functions of different atlastin isoforms .

These findings indicate that the HVR is not merely a linker region but plays active roles in both the enzymatic function of Atlastin-1 and its regulation within the cellular environment.

What are the optimal conditions for expressing and purifying recombinant Mouse Atlastin-1?

Based on established protocols, the following conditions are optimal for expressing and purifying recombinant Mouse Atlastin-1:

Expression System:

• Cell line: HEK293T cells provide a eukaryotic environment that supports proper folding and post-translational modifications .

• Tags: C-terminal MYC/DDK tags facilitate purification while minimizing interference with protein function .

Purification Parameters:

• Buffer composition: 25 mM Tris.HCl, pH 7.3, 100 mM glycine, 10% glycerol provides stability during storage .

• Purity assessment: SDS-PAGE and Coomassie blue staining can verify purity (target >80%) .

Storage Specifications:

• The purified recombinant protein should be stored with 10% glycerol to maintain stability .

• Aliquoting is recommended to avoid freeze-thaw cycles that could compromise protein integrity.

How can researchers effectively assess neurite outgrowth in Atlastin-1 deficient neurons?

To effectively assess neurite outgrowth in Atlastin-1 deficient neurons, researchers should consider the following methodological approach:

• Low-efficiency transfection: Transfect neurons with vectors expressing fluorescent proteins (e.g., eGFP and emerald) 24 hours before fixation to visualize individual neurons . This enables clear delineation of neuronal processes against the background.

• Timing of analysis: Fix neurons at day 5 of differentiation to capture the developing axon stage . At this stage, most neurons have a single process which is likely the developing axon.

• Imaging: Use immunofluorescence microscopy to visualize the transfected neurons and their processes .

• Measurement parameters: Measure the length of the single process (developing axon) to quantify differences between control and Atlastin-1 depleted neurons . Research has shown that on average, the developing axon is significantly longer in neurons depleted of atlastin-1 .

• Statistical analysis: Apply appropriate statistical tests to determine if differences in process length between control and experimental groups are significant.

This approach has successfully demonstrated that atlastin-1 is required for normal morphology of the developing axon in human cortical neurons, with its depletion resulting in longer axonal processes .

What methods can be used to study the conformational changes of Atlastin-1 during GTP hydrolysis?

Several sophisticated methods can be employed to study the conformational changes of Atlastin-1 during GTP hydrolysis:

• X-ray crystallography: This has been successfully used to determine structures of ATL1 at 2.2Å resolution (native protein) and 3.5Å (selenomethionine-derivatized protein) . Different nucleotide-bound states (e.g., GDP-bound) can reveal conformational states during the GTPase cycle.

• Limited proteolysis: This technique can probe the accessibility of proteolytic sites, which changes with protein conformation. It has been used to characterize how mutations affect intramolecular interactions and GTPase-driven conformational changes .

• FRET-based studies: Fluorescence resonance energy transfer can monitor real-time conformational changes in the protein . By strategically placing fluorophores on different domains, researchers can track their relative movements during the GTPase cycle.

• Structural analysis of disease mutations: Characterizing mutations like the N417ins insertion can provide insights into regions critical for conformational change. This particular mutation affects a region central to intramolecular interactions and GTPase-driven conformational change, leading to an aberrant prehydrolysis state .

• Membrane tethering assays: These functional assays can assess how conformational changes translate to biological activity. Some mutations may not affect GTPase activity but can alter membrane tethering efficiency, highlighting the importance of proper conformational dynamics .

These methods collectively provide a comprehensive view of how Atlastin-1 undergoes structural rearrangements during its catalytic cycle, which is essential for its membrane fusion activity.

How can recombinant Atlastin-1 be used to investigate protein-protein interactions?

Recombinant Atlastin-1 provides a valuable tool for investigating protein-protein interactions through several approaches:

• Co-immunoprecipitation studies: Using tagged recombinant Atlastin-1 (such as the C-MYC/DDK-tagged mouse Atl1) , researchers can pull down Atlastin-1 complexes from cell lysates and identify interacting partners through mass spectrometry or western blotting.

• Direct binding assays: Purified recombinant Atlastin-1 can be used in in vitro binding assays with candidate interacting proteins to assess direct interactions.

• Known interaction partners: Studies have shown that Atlastin-1 interacts with spastin and with mitogen-activated protein kinase kinase kinase kinase 4 . Recombinant Atlastin-1 can be used to further characterize these interactions and identify binding domains.

• Homotetramer formation: Atlastin-1 can form a homotetramer , and recombinant protein can be used to study the structural and functional implications of this oligomerization.

• Crystal packing analysis: Analysis of crystal structures has revealed that the HVR of ATL1 makes direct contacts with the G domain of adjacent protomers . This observation suggests a role for the HVR in coordinating the catalytic cycles of multiple ATLs through protein-protein interactions.

Understanding these interactions is crucial for elucidating Atlastin-1's role in ER morphology regulation and identifying potential therapeutic targets for Atlastin-1-related disorders.

What are the implications of Atlastin-1 research for understanding and treating hereditary spastic paraplegia?

Research on Atlastin-1 has several important implications for understanding and potentially treating hereditary spastic paraplegia (HSP):

• Pathogenic mechanisms: Studies have revealed that atlastin-1 deficiency leads to altered ER morphology, increased endosomal tubulation, and reduced lysosomal proteolytic capacity . This suggests multiple cellular pathways through which ATL1 mutations could lead to neurodegeneration in HSP.

• Therapeutic targets: The connection between atlastin-1 dysfunction and lysosomal abnormalities suggests that enhancing lysosomal function might be a therapeutic strategy for ATL1-related HSP .

• Genotype-phenotype correlations: Different types of ATL1 mutations (missense, loss-of-function, gain-of-function) appear to be associated with different disease mechanisms (dominant negative, haploinsufficiency) and potentially different disease severities . This understanding could help predict disease progression based on specific mutations.

• Mutation-specific effects: Detailed characterization of novel mutations, such as the N417ins insertion, reveals how specific structural alterations affect protein function . The N417ins mutation affects a region central to intramolecular interactions and GTPase-driven conformational change, leading to an aberrant prehydrolysis state .

• Neurodevelopmental aspects: Atlastin-1 depletion affects neurite growth but not neuronal viability , suggesting that therapeutic interventions might need to focus on axonal development and maintenance rather than neuronal survival.

These insights highlight the complexity of HSP pathogenesis and suggest that personalized therapeutic approaches targeting specific disease mechanisms might be necessary for effective treatment.

How does Atlastin-1 research contribute to understanding broader neurological disease mechanisms?

Atlastin-1 research provides valuable insights into broader neurological disease mechanisms beyond hereditary spastic paraplegia:

• ER-lysosome crosstalk: The finding that deficiency of a classical ER morphogen such as atlastin-1 causes abnormal lysosomal function strengthens the idea that ER and lysosome functions are linked in neurons . This has implications for various neurodegenerative disorders where lysosomal dysfunction is observed.

• Axonal maintenance: Atlastin-1's role in axonal maintenance provides insights into how long axons are maintained in the nervous system, which is relevant to many neurological conditions involving axonal degeneration.

• Membrane dynamics: The study of atlastin-1's role in membrane fusion and ER morphology enhances our understanding of cellular membrane dynamics, which are important in numerous neurological disorders.

• Protein quality control: The connection between ER morphology and protein degradation pathways (including lysosomal proteolysis) highlights the importance of protein quality control mechanisms in neuronal health and disease.

• Developmental neurobiology: The switch in atlastin isoform expression during neural differentiation provides insights into developmental processes that might be relevant to neurodevelopmental disorders.

By elucidating these fundamental cellular mechanisms, atlastin-1 research contributes to a broader understanding of neuronal homeostasis and how its disruption leads to neurological disease.

What are the most promising areas for future Atlastin-1 research?

Based on current knowledge and gaps in understanding, several promising areas for future Atlastin-1 research emerge:

• Therapeutic modulation: Developing compounds that can enhance atlastin-1 function or compensate for its deficiency in neurons affected by HSP-causing mutations.

• Isoform redundancy: Further exploring the functional redundancy between atlastin isoforms to understand why atlastin-1 mutations cause primarily neurological symptoms despite the presence of other atlastins.

• Regulatory mechanisms: Investigating the physiological significance of post-translational modifications of the HVR and identifying the specific kinases involved in this regulation .

• ER-lysosome communication: Elucidating the molecular mechanisms by which atlastin-1-mediated ER morphology influences lysosomal function .

• Axonal transport: Examining how atlastin-1 deficiency affects axonal transport processes, which might explain the length-dependent axonal degeneration seen in HSP.

• Cell type specificity: Determining why particular neuronal populations (corticospinal tract neurons) are especially vulnerable to atlastin-1 dysfunction.

• Interaction with other HSP proteins: Investigating functional interactions between atlastin-1 and other HSP-associated proteins to identify common pathogenic pathways.