Recombinant Rat Atlastin-1 (Atl1)

What is the molecular structure of rat Atlastin-1 and how does it compare to human Atlastin-1?

Rat Atlastin-1 (Atl1) is a 558-amino acid protein that functions as a dynamin-like GTPase. The full protein sequence includes a GTPase domain at the N-terminus, followed by middle and C-terminal domains . The protein contains critical regions for GTP binding and hydrolysis, with a key lysine residue (K80) in the GTPase domain being essential for its function. When comparing rat and human Atlastin-1, they share high sequence homology, which is why rat models are valuable for studying Atlastin-1-related human diseases .

The protein's structure is characterized by:

• N-terminal GTPase domain (critical for membrane fusion activity)

• Two closely spaced transmembrane domains

• C-terminal cytosolic domain

Mutations that disrupt GTPase activity, such as K80A (equivalent to K51A in some numbering systems), eliminate the protein's ability to mediate membrane fusion and establish proper ER networks .

What are the primary functions of Atlastin-1 in neuronal cells?

Atlastin-1 plays several crucial roles in neuronal cells:

• ER Morphogenesis: Atlastin-1 mediates homotypic membrane fusion to establish endoplasmic reticulum networks, particularly at dendrite branch points and in the neuronal soma .

• Dendritic ER Maintenance: It ensures proper extension of ER tubules into dendritic branches, which is critical for dendritic stability and function .

• Mitochondrial Regulation: Atlastin-1 influences mitochondrial fission at dendritic branch points, indicating its role in coordinating ER-mitochondria interactions .

• Synaptic Function: Studies suggest that Atlastin-1 affects synaptic vesicle pools and neurotransmitter release. In particular, Atlastin-1 overexpression has been shown to increase inhibitory synaptic transmission, suggesting a role in regulating neuronal excitability .

• Protein Homeostasis: Atlastin-1 appears to play a role in the unfolded protein response (UPR), contributing to proper protein folding and cellular stress responses .

Disruption of these functions through mutations or altered expression levels can lead to neuronal pathologies, including Hereditary Spastic Paraplegia and potentially contribute to epilepsy .

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

For successful expression and purification of recombinant rat Atlastin-1:

Expression System:

• E. coli is commonly used for full-length rat Atlastin-1 expression with N-terminal His-tag .

• The full-length protein (amino acids 1-558) can be successfully expressed and purified for functional studies .

Purification Protocol:

• Express His-tagged full-length rat Atlastin-1 in E. coli

• Harvest cells and lyse in Tris/PBS-based buffer

• Purify using nickel affinity chromatography

• Elute with imidazole

• Dialyze against Tris/PBS buffer at pH 8.0

• Lyophilize for long-term storage

Storage Conditions:

• Store lyophilized protein at -20°C/-80°C

• For working solutions, reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Add glycerol (recommended final concentration 50%) for long-term storage

• Avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

The purity of properly prepared recombinant rat Atlastin-1 should exceed 90% as determined by SDS-PAGE analysis .

How can researchers effectively assess the GTPase activity of recombinant Atlastin-1?

Evaluating the GTPase activity of Atlastin-1 is critical for functional studies, particularly when investigating disease-associated mutations. Several complementary approaches can be employed:

In vitro GTPase Assays:

• Radioactive GTP Hydrolysis Assay: Measure the conversion of [γ-32P]GTP to GDP and inorganic phosphate

• Colorimetric Phosphate Release Assay: Using malachite green to detect inorganic phosphate released during GTP hydrolysis

• HPLC-based Methods: To separate and quantify GTP and GDP

Functional Membrane Fusion Assays:

• Liposome Fusion Assay: Reconstitute purified Atlastin-1 into liposomes and measure fusion rates

• ER Membrane Fusion in Cell-free Systems: Using isolated ER vesicles to assess fusion capacity

Cellular GTPase Function Assessment:

• ER Morphology Analysis: Examine ER network formation in COS-7 cells or similar models

• Three-way Junction Quantification: Measure the density of 3-way junctions in ER networks as a readout of functional GTPase activity

When analyzing disease-associated mutations like P183L (equivalent to P219L in some numbering systems), researchers have demonstrated these mutations dramatically reduce GTPase activity and impair membrane tethering and fusion capacity, similar to the known GTP-binding defective K51A mutation .

What experimental models are most appropriate for studying Atlastin-1 function in neurons?

Several experimental models have proven effective for investigating Atlastin-1 function in neurons:

In vitro Models:

• Primary Neuron Cultures: Rat or mouse primary neurons allow for detailed analysis of ER morphology, protein localization, and electrophysiological properties

• Neuronal Cell Lines: Such as differentiated PC12 cells or SH-SY5Y cells for preliminary studies

In vivo Models:

• C. elegans PVD Neuron Model: Particularly valuable for studying dendritic ER morphology due to its stereotyped dendritic branching pattern. This model has been instrumental in identifying ATLN-1's role in establishing ER networks in dendrites .

• Mouse Models:

  • Pentylenetetrazol (PTZ)-kindled epileptic mouse models for studying Atlastin-1's role in seizure activity

  • Transgenic mice expressing disease-associated Atlastin-1 mutations

• Drosophila Models: Useful for examining Atlastin function in motor neurons and at synaptic terminals

Experimental Techniques:

• Lentiviral Expression Systems: For overexpression or knockdown of Atlastin-1 in specific neuronal populations

• Patch-clamp Recordings: To assess neuronal excitability and synaptic transmission following Atlastin-1 manipulation

• Live Cell Imaging: For visualizing ER dynamics in dendrites and axons

• ER Markers: Using various ER-targeted fluorescent proteins to visualize ER morphology in neuronal compartments

The C. elegans PVD neuron has been particularly informative, revealing that ATLN-1 is required for proper ER network formation at dendrite branch points and for extending ER tubules into high-order dendritic branches .

How do mutations in Atlastin-1 contribute to Hereditary Spastic Paraplegia pathogenesis?

Atlastin-1 mutations represent one of the genetic causes of Hereditary Spastic Paraplegia (HSP), with specific mechanisms:

ER Morphology Disruption:
Mutations in Atlastin-1 lead to abnormal ER morphology characterized by:

• Reduced complexity of ER networks

• Fewer three-way junctions in the ER

• Increased unbranched, parallel ER tubules

• Retraction of ER tubules from high-order dendritic branches

These structural changes disrupt the normal function of neuronal ER, particularly in dendrites and at branch points.

Functional Consequences:

• Destabilized Microtubules: Abnormal ER morphology in Atlastin-1 mutants causes destabilization of microtubules in dendritic branches

• Mitochondrial Dysfunction: Defective ER morphology leads to impaired mitochondrial fission at dendritic branch points

• Protein Homeostasis Impairment: Abnormal ER function likely contributes to defects in protein synthesis and folding, particularly when unfolded protein response (UPR) is compromised

Types of Pathogenic Mutations:
Most Atlastin-1 mutations found in HSP patients are heterozygous and can function as dominant-negative alleles, further inhibiting the function of the wild-type allele. Studies comparing different mutations found in HSP patients show similar ER phenotypes, strongly suggesting that neuronal ER impairment is a key contributor to HSP disease pathogenesis .

What is the relationship between Atlastin-1 and epilepsy?

Recent research has revealed an unexpected relationship between Atlastin-1 and epilepsy:

Expression Changes in Epilepsy:

• Atlastin-1 protein expression is reduced in the temporal neocortex of patients with temporal lobe epilepsy

• Similar reduction is observed in the hippocampus and adjacent cortex of pentylenetetrazol (PTZ)-kindled epileptic mouse models

Cellular Localization:

• Cells expressing Atlastin-1 coexpress GAD67, an inhibitory synaptic marker, in both human and mouse epileptic tissues

• This suggests Atlastin-1 may play a role specifically in inhibitory neurotransmission

Functional Effects on Seizure Activity:

• Lentivirus-mediated overexpression of Atlastin-1 in the hippocampus suppresses seizure activity in behavioral experiments

• This anticonvulsant effect appears to be mediated through modulation of neuronal excitability

Electrophysiological Mechanisms:

• Atlastin-1 overexpression inhibits neuronal excitability by suppressing the discharge frequency of spontaneous action potentials

• Notably, Atlastin-1 increases inhibitory synaptic transmission without affecting excitatory synaptic currents

• These findings suggest Atlastin-1 contributes to epilepsy pathogenesis through modulation of inhibitory synaptic transmission

This evidence points to Atlastin-1 as a potential therapeutic target for epilepsy, particularly for approaches aimed at enhancing inhibitory neurotransmission to restore excitatory/inhibitory balance.

What are the critical controls when assessing Atlastin-1 function in membrane fusion assays?

When designing and interpreting membrane fusion assays for Atlastin-1, several critical controls should be included:

Negative Controls:

• GTPase-deficient Mutants: The K80A mutation (disrupting GTP binding) serves as an essential negative control, as it eliminates fusion activity while maintaining protein expression

• GTP-free Conditions: Assays performed without GTP addition

• Non-fusogenic Proteins: Including unrelated membrane proteins without fusion activity

Positive Controls:

• Wild-type Atlastin-1: Full-length protein with intact GTPase domain

• Known Fusogenic Proteins: Other well-characterized membrane fusion proteins (e.g., SNARE proteins)

Domain-specific Controls:

• Isolated GTPase Domain: To assess GTP binding and hydrolysis independent of fusion

• Transmembrane Domain Mutants: To distinguish between GTPase activity and membrane insertion defects

Experimental Variables to Control:

• Protein:Lipid Ratio: Different ratios can significantly impact fusion efficiency

• Membrane Composition: Lipid composition affects Atlastin-1 function

• Temperature and pH: Optimize and maintain consistent conditions

When interpreting results from mutant versions (like the HSP-associated P183L mutation), compare both GTPase activity and membrane fusion capability to distinguish between primary defects in enzymatic activity versus secondary effects on fusion .

How should researchers interpret contradictory findings regarding Atlastin-1's role in different neuronal compartments?

The literature contains some apparent contradictions regarding Atlastin-1's role in different neuronal compartments (axons vs. dendrites). To properly interpret these findings:

Compartment-specific Considerations:

• Axonal vs. Dendritic ER:

  • While most literature has focused on Atlastin-1's function in axonal ER, evidence shows it also plays crucial roles in dendritic ER morphology

  • In Drosophila motor neurons, Atlastin manipulation affects axon terminals, while in mammalian neurons and C. elegans, effects on dendritic ER are prominent

  • These differences likely reflect compartment-specific ER organization and function

• Model System Variations:

  • Different model systems show varying phenotypes

  • C. elegans PVD neurons show clear effects on dendritic ER

  • Drosophila studies emphasize axonal ER effects

  • Mammalian cortical neurons show effects on dendritic growth and branching

Reconciling Contradictions:

• Consider developmental timing: Effects may vary at different developmental stages

• Distinguish between overexpression and loss-of-function: Overexpression of wild-type Atlastin-1 versus GTPase-dead forms may have opposite effects

• Evaluate cell-type specificity: Effects may differ between neuron types

• Assess compensation by paralogs: Other Atlastin family members may compensate in specific compartments

The apparent contradictions likely reflect the complex roles of Atlastin-1 in different neuronal compartments rather than true inconsistencies. A comprehensive model would recognize that Atlastin-1 functions in both axonal and dendritic ER, but with compartment-specific requirements and effects that vary by neuron type and model system .

What technical challenges are associated with studying Atlastin-1 in primary neuronal cultures?

Researchers face several technical challenges when studying Atlastin-1 in primary neuronal cultures:

Protein Expression and Delivery:

• Transfection Efficiency: Primary neurons are notoriously difficult to transfect with standard methods

  • Solution: Use lentiviral or AAV vectors for higher efficiency

  • Alternative: Nucleofection prior to plating for moderate efficiency

• Expression Level Control: Overexpression may cause non-physiological effects

  • Solution: Use inducible expression systems or promoters with moderate strength

  • Control: Always compare to endogenous expression levels

Visualization Challenges:

• ER Morphology Assessment: The complex architecture of neuronal ER makes detailed visualization difficult

  • Solution: Use super-resolution microscopy techniques

  • Control: Compare multiple ER markers to ensure consistent observations

• Compartment-specific Analysis: Distinguishing ER in different neuronal compartments

  • Solution: Use microfluidic chambers to isolate axons and dendrites

  • Alternative: Sparse labeling techniques to visualize individual neurons fully

Functional Assessments:

• Electrophysiology and Atlastin-1: Establishing causality between Atlastin-1 manipulation and electrophysiological changes

  • Solution: Use acute manipulation systems (optogenetics/chemogenetics)

  • Control: Include wild-type and GTPase-deficient mutants for comparison

• Temporal Dynamics: Capturing dynamic ER remodeling

  • Solution: Live-cell imaging with minimal phototoxicity

  • Control: Time-matched controls for all experiments

Troubleshooting Strategies:

• For poor viral transduction: Optimize virus titer and incubation time

• For toxicity: Reduce expression levels and confirm cell health markers

• For inconsistent ER visualization: Try multiple ER markers and fixation protocols

• For patch-clamp recording difficulties: Standardize recording conditions and cell selection criteria

How can researchers effectively use Atlastin-1 antibodies for protein detection and localization studies?

For optimal use of Atlastin-1 antibodies in research:

Antibody Selection:
Polyclonal antibodies like 12149-1-AP have demonstrated reactivity with human, mouse, and rat Atlastin-1 in various applications including Western blot (WB) and Co-immunoprecipitation (CoIP) .

Optimization for Western Blotting:

• Expected Molecular Weight: ~64 kDa for full-length Atlastin-1

• Sample Preparation:

  • Tissue lysates: Use RIPA buffer with protease inhibitors

  • Cell lysates: Add phosphatase inhibitors if examining phosphorylation status

• Controls:

  • Positive control: Recombinant Atlastin-1 protein

  • Negative control: Lysates from Atlastin-1 knockout cells

Immunocytochemistry Considerations:

• Fixation: 4% paraformaldehyde (10-15 minutes) works well for ER proteins

• Permeabilization: 0.1% Triton X-100 is sufficient

• Co-staining: Combine with ER markers (calnexin, PDI) for colocalization analysis

• Controls: Include peptide competition assays to confirm specificity

Co-immunoprecipitation Protocols:

• Lysis Conditions: Use mild detergents (0.5-1% NP-40 or CHAPS) to preserve protein-protein interactions

• Pre-clearing: Always pre-clear lysates with appropriate control IgG

• Positive Controls: Include known Atlastin-1 interaction partners

• Detection: Use clean detection antibodies from different host species

Troubleshooting Common Issues:

• High Background: Increase blocking time or detergent in wash buffers

• Multiple Bands: Validate with recombinant protein control

• Weak Signal: Optimize antibody concentration or increase protein loading

• Non-specific Binding: Use more stringent washing conditions

For subcellular localization studies, compare antibody staining patterns with fluorescently tagged Atlastin-1 to confirm specificity and avoid fixation artifacts that can distort ER morphology .

What are promising research directions for understanding Atlastin-1's role in neurological disorders beyond HSP?

Based on recent findings, several promising research directions emerge for understanding Atlastin-1's broader roles in neurological disorders:

Epilepsy Mechanisms:

• Further investigate how Atlastin-1 modulates inhibitory neurotransmission

• Explore whether Atlastin-1 expression changes are cause or consequence of epileptic activity

• Develop targeted approaches to enhance Atlastin-1 function in inhibitory neurons as potential therapeutic strategy

Neurodevelopmental Disorders:

• Examine Atlastin-1's role in dendritic development and synaptogenesis

• Investigate potential relationships with autism spectrum disorders or intellectual disability

• Study the impact of Atlastin-1 mutations on neuronal circuit formation during development

ER-Mitochondria Interactions:

• Further characterize how Atlastin-1 regulates mitochondrial fission and function at dendritic branch points

• Investigate links to mitochondrial dysfunction in neurodegenerative disorders

• Explore potential roles in calcium homeostasis at ER-mitochondria contact sites

Protein Homeostasis and Neurodegeneration:

• Examine Atlastin-1's contribution to protein quality control and the unfolded protein response

• Investigate connections to protein aggregation diseases (Alzheimer's, Parkinson's)

• Study how Atlastin-1 dysfunction affects neuronal proteostasis during aging

Therapeutic Approaches:

• Small Molecule Modulators: Develop compounds that enhance Atlastin-1 GTPase activity

• Gene Therapy: Test viral-mediated delivery of Atlastin-1 in disease models

• Downstream Pathway Targeting: Identify and modulate key pathways affected by Atlastin-1 dysfunction

These research directions could expand our understanding of Atlastin-1 beyond its established role in HSP and potentially identify new therapeutic targets for multiple neurological disorders .

What are the most reliable quantitative methods for analyzing ER morphology defects in Atlastin-1 mutants?

Reliable quantification of ER morphology defects is crucial for studying Atlastin-1 function:

ER Network Complexity Metrics:

• Three-way Junction Density: Quantify the number of three-way junctions per unit area, a key indicator of network complexity that is directly affected by Atlastin-1 function

• Tubule Length Distribution: Measure the length of individual ER tubules between junctions

• Network Polygon Analysis: Assess the size and shape of polygons formed by ER networks

Dendritic ER Analysis:

• ER Extension into Branches: Measure the distance ER tubules extend into dendritic branches of different orders

• ER Continuity Assessment: Quantify fragmentation of ER networks along dendrites

• Branch Point ER Complexity: Specifically analyze ER morphology at branch points where Atlastin-1 is particularly important

Image Analysis Approaches:

• Semi-automated Tracing: Software like NeuronJ or Simple Neurite Tracer for initial ER tubule detection

• Machine Learning Algorithms: Trained on ER morphology datasets for automated segmentation

• 3D Reconstruction: For volumetric analysis of ER networks from confocal z-stacks

Statistical Analysis:

• Compare multiple parameters between wild-type and mutant conditions

• Use appropriate statistical tests (t-test, ANOVA with post-hoc tests)

• Include multiple cells from multiple independent experiments

• Consider blinded analysis to avoid bias

When studying C. elegans PVD neurons, researchers have successfully quantified ER extension into different branch orders, revealing that in Atlastin-1 mutants, ER tubules retract from high-order dendritic branches. This approach directly ties structural ER defects to specific functional domains of neuronal dendrites .

How should electrophysiological data be interpreted when studying Atlastin-1's effects on neuronal excitability?

When analyzing electrophysiological data related to Atlastin-1 function:

Action Potential Parameters:

Synaptic Transmission Analysis:

• Inhibitory vs. Excitatory Currents: Separate analysis is crucial, as Atlastin-1 overexpression specifically increases inhibitory synaptic transmission without affecting excitatory synaptic currents

• mIPSCs vs. sIPSCs: Distinguish between miniature (action potential-independent) and spontaneous (includes action potential-driven) events to determine pre- versus post-synaptic effects

• Paired-pulse Ratios: Assess changes in presynaptic release probability

Data Interpretation Framework:

• Compare multiple parameters to build a comprehensive picture of Atlastin-1's effects

• Consider both direct effects (on membrane properties) and indirect effects (on synaptic transmission)

• Correlate electrophysiological findings with morphological observations

• Compare effects in different neuron types (excitatory vs. inhibitory)

Experimental Models:
For epilepsy research, the Mg²⁺-free epilepsy cell model has proven valuable, enabling researchers to demonstrate that Atlastin-1 overexpression inhibits neuronal excitability by suppressing action potential frequency rather than by changing passive or active properties of action potentials themselves .

This analytical approach has revealed that Atlastin-1 likely contributes to epilepsy through effects on inhibitory synaptic transmission, providing a more nuanced understanding of its neurophysiological functions beyond ER morphology .

What are the key considerations for designing functional studies with recombinant rat Atlastin-1?

When designing functional studies with recombinant rat Atlastin-1:

Protein Design Considerations:

• Tag Selection: His-tags are commonly used and don't significantly interfere with function , but consider:

  • N-terminal vs. C-terminal tags (N-terminal generally preferred for Atlastin-1)

  • Tag size (smaller tags minimize functional interference)

  • Cleavable tags for removal post-purification

• Domain Structure: Full-length protein (1-558aa) includes all functional domains , but truncated constructs may be useful:

  • Isolated GTPase domain for enzymatic studies

  • Cytoplasmic domain for structural analysis

  • Transmembrane domain-deleted versions for solubility

• Mutation Design: Strategic mutations provide valuable functional insights:

  • K80A: Disrupts GTP binding, serves as negative control

  • P183L/P219L: Disease-relevant mutation found in HSP patients

  • Site-directed mutations at key residues for structure-function analysis

Experimental Applications:

• In vitro Reconstitution: Recombinant Atlastin-1 can be reconstituted into liposomes to study membrane fusion

• GTPase Assays: Purified protein enables direct measurement of enzymatic activity

• Protein-Protein Interaction Studies: Pull-down assays to identify binding partners

• Structural Studies: Crystallography or cryo-EM analysis of protein structure

Controls and Validations:

• Functional Validation: Verify GTPase activity of purified protein

• Structural Integrity: Circular dichroism or limited proteolysis to confirm proper folding

• Oligomerization Assessment: Size exclusion chromatography to verify dimerization capacity

• Activity Comparisons: Always compare with human ortholog when studying disease-relevant mutations

For optimized experimental design, consider that recombinant Atlastin-1 is typically stored in Tris/PBS-based buffer (pH 8.0) with 6% trehalose and can be reconstituted to 0.1-1.0 mg/mL in deionized sterile water with added glycerol for long-term storage .

How do the functional properties of recombinant rat Atlastin-1 compare with human and mouse orthologs?

Understanding cross-species comparisons is essential for translational research:

Sequence Homology and Conservation:

• Rat Atlastin-1 shares high sequence similarity with both mouse and human orthologs

• The GTPase domain is particularly well-conserved across species

• Key functional residues, including the critical lysine in the GTPase domain (K80) and proline residues implicated in HSP (P183/P219), are conserved

Functional Conservation:

• GTPase Activity: Core enzymatic function is preserved across species

• Membrane Fusion Capacity: All orthologs mediate homotypic ER membrane fusion

• Oligomerization Properties: Dimerization mechanism appears conserved

Species-Specific Differences:

• Post-translational Modifications: May vary between species

• Tissue Expression Patterns: Some differences in expression levels across tissues

• Interaction Partners: Minor variations in protein-protein interactions

Experimental Cross-Reactivity:

• Antibodies: Some antibodies like 12149-1-AP show cross-reactivity with human, mouse, and rat Atlastin-1

• Functional Assays: Recombinant proteins from different species can generally be used in the same assay systems

Disease Model Relevance:

• Rat and mouse Atlastin-1 serve as good models for human disease-causing mutations

• Introducing equivalent mutations (like P219L in rat corresponding to P208L in human) produces similar functional defects

• This conservation validates the use of rodent models for studying HSP-associated Atlastin-1 mutations