Recombinant Drosophila persimilis Spastin (spas)

What is Drosophila Spastin and what is its biological function?

Spastin is a conserved microtubule (MT)-severing protein involved in cytoskeletal rearrangement processes. It plays critical roles in multiple cellular functions requiring coordination between cytoskeleton rearrangement and membrane remodeling, including neurite branching, axonal growth, midbody abscission, and endosome tubulation . Spastin belongs to the AAA (ATPases Associated with diverse cellular Activities) ATPase family and is closely related in sequence to the microtubule-severing protein Katanin . In its catalytically active form, Spastin severs microtubules, enabling proper regulation of microtubule dynamics in neurons .

Why is Drosophila Spastin used as a model for human Spastin-related disorders?

Drosophila Spastin serves as an excellent model for studying human Spastin-related disorders due to strong functional conservation between species. Human patients with mutations in the SPAST gene develop autosomal dominant hereditary spastic paraplegia (AD-HSP), characterized by progressive weakness and spasticity of the lower limbs. Remarkably, Drosophila lacking spastin exhibit behavioral similarities to human AD-HSP patients . Functional conservation is further demonstrated by the ability of both wild-type Drosophila and human spastin to rescue behavioral and cellular defects in spastin null flies equivalently . This conservation allows researchers to create genetically representative models of AD-HSP in Drosophila, enabling investigation of disease mechanisms and potential therapeutic approaches .

What are the key properties of Recombinant Drosophila persimilis Spastin?

Recombinant Drosophila persimilis Spastin (spas) is typically produced in mammalian cell expression systems with purity levels >85% as determined by SDS-PAGE . The protein has enzymatic activity (EC= 3.6.4.3) and requires proper storage conditions for maintaining stability . It's available in both liquid and lyophilized forms, with shelf lives of approximately 6 months and 12 months at -20°C/-80°C, respectively . For optimal use in experimental applications, reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL is recommended, with addition of 5-50% glycerol for long-term storage .

How should Recombinant Drosophila persimilis Spastin be handled and stored for optimal activity?

For optimal handling and storage of Recombinant Drosophila persimilis Spastin:

• Initial Processing: Briefly centrifuge the vial before opening to bring contents to the bottom .

• Reconstitution: Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Long-term Storage: Add glycerol to a final concentration of 5-50% (with 50% being recommended) and aliquot for storage at -20°C/-80°C .

• Working Aliquots: Store at 4°C for up to one week .

• Avoid Freeze-Thaw Cycles: Repeated freezing and thawing is not recommended as it may compromise protein activity .

The shelf life varies depending on storage form and conditions: liquid preparations typically remain stable for 6 months at -20°C/-80°C, while lyophilized forms maintain stability for approximately 12 months .

What are the recommended approaches for studying Spastin function in Drosophila models?

Several robust approaches have been established for studying Spastin function in Drosophila:

• Genetic Manipulation:

  • Null mutations: Complete deletion of endogenous spastin gene to study loss-of-function effects .

  • Hypomorphic alleles: Partial reduction in Spastin function, producing milder phenotypes .

  • Transgenic expression: Introduction of wild-type or mutant Spastin variants (either Drosophila or human) to assess rescue capabilities or dominant effects .

• Behavioral Assays:

  • Climbing assays: Quantitative measurement of motor function deficits .

  • Flight and jumping tests: Assessment of complex motor behaviors impaired in spastin mutants .

  • Longevity assessments: Tracking lifespan changes associated with Spastin mutations .

• Cellular/Subcellular Analysis:

  • Neuromuscular junction (NMJ) morphology examination: Analysis of synaptic bouton number, clustering, and distribution .

  • Microtubule network visualization: Assessment of cytoskeletal organization in neurons and muscles .

  • Electrophysiological recordings: Measurement of synaptic transmission efficiency .

These complementary approaches provide comprehensive insights into Spastin function at molecular, cellular, and organismal levels.

How can researchers effectively compare the functions of Drosophila and human Spastin in experimental systems?

To effectively compare Drosophila and human Spastin functions:

• Transgenic Rescue Experiments: Express human Spastin variants in Drosophila spastin null backgrounds and quantify the degree of phenotypic rescue at behavioral, cellular, and subcellular levels . This approach allows direct assessment of functional conservation and disease-causing mutations.

• Parallel Expression Systems: Generate equivalent mutations in both Drosophila and human Spastin and express them in identical cellular contexts to compare biochemical and cellular effects .

• Domain Swap Experiments: Create chimeric proteins containing domains from both Drosophila and human Spastin to identify functionally conserved regions.

• Quantitative Phenotypic Assessment: Develop standardized assays to measure:

  • Eclosion rates (fly development to adulthood)

  • Motor behavior metrics

  • Synaptic morphology parameters

  • Microtubule organization patterns

For example, pan-neuronal expression of fly wild-type spastin (D WT) in a null background significantly rescues eclosion rates from <6% to approximately 50% . Human wild-type Spastin demonstrates comparable rescue efficiency, confirming functional conservation between species .

How do different mutations in Spastin affect its function in Drosophila models?

Different Spastin mutations produce distinct functional consequences in Drosophila models:

• Complete Loss-of-Function (Null) Mutations:

  • Severe behavioral deficits: Inability to fly or jump, poor climbing ability, and shortened lifespan .

  • NMJ abnormalities: Increased number and clustering of synaptic boutons .

  • Impaired neurotransmitter release .

  • Reduced microtubule bundles within NMJs, particularly in distal boutons .

• Hypomorphic Mutations (Partial Function):

  • Milder behavioral phenotypes compared to null mutants .

  • Intermediate NMJ abnormalities.

• Catalytic Domain Mutations (e.g., K388R equivalent):

  • When expressed in trans with wild-type Spastin, causes aberrant distal synapse morphology and microtubule distribution, similar to but less severe than nulls .

  • Acts dominantly but provides partial rescue of null phenotypes, suggesting additional non-catalytic Spastin functions .

• N-terminal Modifier Mutations (e.g., S44L and P45Q equivalents):

  • Largely silent when heterozygous .

  • Exacerbate catalytic domain mutation phenotypes when expressed in trans, matching observations from human pedigrees .

These mutation effects provide valuable insights into structure-function relationships and pathogenic mechanisms relevant to human disease.

What is the significance of the paradoxical microtubule phenotype in spastin mutants?

The paradoxical microtubule phenotype in spastin mutants represents an intriguing scientific puzzle that challenges simple models of Spastin function:

Since Spastin functions as a microtubule-severing protein, one would predict that spastin loss-of-function mutants should exhibit an increased number of microtubules due to reduced severing activity. Indeed, overexpression of Spastin completely erases the muscle microtubule network, consistent with its severing function .

This paradox highlights the complexity of cytoskeletal regulation and suggests that severing activity may actually promote microtubule network formation in specific developmental and cellular contexts.

How does Spastin's interaction with lipid droplets relate to its neuronal functions?

The relationship between Spastin's interaction with lipid droplets and its neuronal functions represents an emerging area of investigation with several key implications:

Spastin-M1 (the longer isoform initiated at the first methionine) can sort from the endoplasmic reticulum (ER) to pre- and mature lipid droplets (LDs) . A hydrophobic motif comprising amino acids 57-86 is sufficient for this LD targeting, with arginine 65 playing a critical role .

This interaction affects lipid metabolism in several ways:

• Increased levels of spastin-M1 reduce LD number but increase their size .

• Expression of catalytically inactive Spastin causes LD clustering .

• Ubiquitous overexpression of Dspastin in Drosophila leads to fewer but larger LDs in fat bodies and increased triacylglycerol levels .

• Tissue-specific effects exist: Dspastin overexpression increases LD number in skeletal muscles and nerves .

The functional significance of this relationship may include:

• Potential metabolic basis for axonal degeneration: Disruption of lipid metabolism in neurons with compromised Spastin function may contribute to the axonal pathology observed in hereditary spastic paraplegia.

• Role in energy homeostasis: Spastin may coordinate energy storage/utilization with cytoskeletal dynamics in neurons, which have high energy demands.

• Organelle interactions: Spastin might facilitate interactions between the ER, lipid droplets, and microtubules at specialized cellular domains.

Further investigation of these connections may reveal novel therapeutic avenues targeting metabolic pathways in spastin-related disorders.

What methodological approaches are recommended for analyzing the effects of Spastin on microtubule networks?

For comprehensive analysis of Spastin's effects on microtubule networks, researchers should employ multifaceted methodological approaches:

• Live Imaging Techniques:

  • Fluorescently tagged tubulin expression for real-time visualization of microtubule dynamics

  • FRAP (Fluorescence Recovery After Photobleaching) analysis to measure microtubule turnover rates

  • Super-resolution microscopy for detailed microtubule network architecture

• Biochemical Assays:

  • In vitro microtubule severing assays using purified components

  • ATPase activity measurements to correlate enzymatic function with severing activity

  • Co-sedimentation assays to quantify microtubule-binding properties

• Quantitative Analysis Parameters:

  • Microtubule bundle density and distribution

  • Microtubule stability (acetylated vs. tyrosinated tubulin ratios)

  • Growth/shrinkage rates of dynamic microtubules

  • Branching frequency and network complexity

• Model System Considerations:

  • Primary neuronal cultures allow detailed analysis of axonal and dendritic microtubules

  • Drosophila neuromuscular junction preparations enable examination of synaptic microtubule organization

  • Muscle cells provide large-scale cytoskeletal networks suitable for gain-of-function experiments

When interpreting results, researchers should be particularly attentive to the paradoxical finding that loss of Spastin results in fewer organized microtubule bundles within neuromuscular junctions , suggesting complex regulation beyond simple severing activity.

How can researchers effectively model and study human Spastin mutations using Drosophila systems?

To effectively model human Spastin mutations using Drosophila systems:

• Generate Genetically Representative Models:

  • Create a modular system using flies deleted for endogenous spastin and expressing human Spastin variants

  • Express human Spastin variants in allelic combinations that mimic human genotypes

  • Ensure the genetic background is consistent across all experimental conditions

• Model Specific Mutation Types:

  • Catalytic domain mutations (e.g., K388R): Express in heterozygous state with wild-type Spastin to model dominant effects

  • Modifier mutations (e.g., S44L, P45Q): Test in trans with catalytic domain mutations to assess interaction effects

  • Haploinsufficiency: Express single-copy wild-type Spastin in null background to model reduced dosage

• Quantification Approaches:

  • Developmental parameters: Eclosion rates (emerging from pupal cases)

  • Behavioral assays: Climbing ability, flight performance, lifespan

  • Cellular phenotypes: NMJ morphology, synaptic bouton number and distribution

  • Subcellular features: Microtubule organization, synaptic vesicle distribution

• Translational Considerations:

  • Compare phenotypic severity across genotypes to establish hierarchy matching human clinical data

  • Test potential therapeutic compounds in established models

  • Validate findings using mammalian cellular models when possible

This approach has successfully recapitulated human disease features, demonstrating that allelic combinations mimicking human genotypes show phenotypic severity conforming to predictions based on human data .

What are the latest insights into the non-canonical functions of Spastin beyond microtubule severing?

Recent research has revealed several non-canonical functions of Spastin beyond its established role in microtubule severing:

• Lipid Metabolism Regulation:

  • Spastin-M1 targets lipid droplets through a hydrophobic motif (amino acids 57-86)

  • Alters lipid droplet size, number, and distribution in tissue-specific patterns

  • Impacts triacylglycerol levels, suggesting a role in lipid storage regulation

• Alternative Structural Functions:

  • Evidence suggests Spastin may have non-catalytic roles, as certain mutations in the catalytic domain still provide partial functional rescue

  • May serve as a structural scaffold for organizing cellular components

• Membrane Remodeling:

  • Participates in processes requiring coordination between cytoskeletal rearrangement and membrane dynamics

  • Involved in endosome tubulation, suggesting roles in membrane trafficking

• Tissue-Specific Functions:

  • Differential effects observed in various tissues (nervous system, muscle, fat bodies)

  • Opposite effects on lipid droplet numbers when expressed in different tissues suggests context-dependent roles

• Genetic Interaction Networks:

  • Functions as both a primary effector and a modifier of other genetic pathways

  • S44L and P45Q variants act as trans-modifiers of catalytic domain mutations

These non-canonical functions may explain why simple models of Spastin function cannot fully account for the complex phenotypes observed in human patients and animal models. They also suggest potential new therapeutic approaches targeting these alternative pathways in Spastin-related disorders.

What common challenges arise when working with Recombinant Drosophila persimilis Spastin and how can they be addressed?

When working with Recombinant Drosophila persimilis Spastin, researchers frequently encounter several challenges:

Additionally, when designing transgenic rescue experiments, it's important to note that complete rescue is often not achieved through pan-neuronal expression, likely because drivers do not precisely mimic the spatiotemporal pattern of endogenous Spastin expression rather than due to insufficient expression levels .

How should researchers interpret apparently contradictory data regarding Spastin function?

When confronted with apparently contradictory data regarding Spastin function, researchers should consider several interpretive frameworks:

• Context-Dependent Activities:

  • Tissue-specific effects: Spastin overexpression increases lipid droplet numbers in muscles and nerves while decreasing them in fat bodies

  • Developmental stage variation: Effects may differ between developing and mature systems

  • Concentration-dependent outcomes: Different levels of Spastin activity may produce opposite phenotypes

• Multiple Functional Domains:

  • Catalytic versus non-catalytic roles: Some mutations affecting the catalytic domain still provide partial rescue, suggesting additional functions

  • Isoform-specific effects: The M1 isoform specifically targets lipid droplets, while both M1 and M87 sever microtubules

• Indirect Effects and System Compensation:

  • The paradoxical reduction in microtubule bundles in spastin-null mutants may reflect compensatory mechanisms or indirect effects on microtubule stability and organization

  • Long-term adaptations to altered Spastin function may mask or transform primary phenotypes

• Methodological Considerations:

  • Different analytical techniques may capture distinct aspects of complex phenotypes

  • Acute versus chronic manipulations may yield different outcomes

When reporting seemingly contradictory findings, researchers should carefully document experimental conditions, genetic backgrounds, and analytical methods to facilitate interpretation and reproducibility.

What are the key considerations for designing experiments to test the effects of potential therapeutic compounds on Spastin-related phenotypes?

When designing experiments to test potential therapeutic compounds for Spastin-related disorders, researchers should address these key considerations:

• Model Selection and Validation:

  • Choose appropriate genetic models representing specific human mutations rather than simple null backgrounds

  • Validate models by confirming that phenotypic severity matches predictions from human clinical data

  • Include both heterozygous and homozygous conditions to capture dominant and recessive effects

• Endpoint Selection:

  • Utilize multi-level analysis spanning behavioral, cellular, and molecular phenotypes

  • Prioritize phenotypes with clear relevance to human pathology (motor function, axon terminal maintenance)

  • Include both early developmental and adult-onset manifestations

• Experimental Design Principles:

  • Implement blinded assessment protocols to eliminate bias

  • Include positive controls (known genetic rescues) and negative controls

  • Use dose-response paradigms to establish therapeutic windows

  • Test acute versus chronic administration regimens

• Phenotypic Assessment Methods:

  • Quantitative behavioral assays: climbing rate, movement velocity, endurance measures

  • NMJ morphology analysis: bouton number, size, distribution patterns

  • Microtubule organization: bundle density, particularly in distal synaptic boutons

  • Lipid metabolism parameters: triacylglycerol levels, lipid droplet characteristics

• Translational Considerations:

  • Assess effects on both developmental and maintenance phenotypes

  • Test compounds in multiple genetic backgrounds to identify genotype-specific responses

  • Evaluate potential side effects on wild-type animals

  • Consider the blood-brain barrier penetration requirements for CNS-targeted therapeutics

This systematic approach enables rigorous evaluation of therapeutic potential while accounting for the complex, multi-faceted nature of Spastin-related pathologies.

What are promising new approaches for studying the relationship between Spastin function and lipid metabolism?

Several promising approaches are emerging to investigate the relationship between Spastin function and lipid metabolism:

• Advanced Imaging Techniques:

  • Correlative light and electron microscopy to visualize Spastin-lipid droplet interactions at ultrastructural level

  • Live imaging of tagged Spastin isoforms and lipid droplets to track dynamic interactions

  • FRET-based sensors to detect Spastin conformational changes upon lipid binding

• Multi-omics Integration:

  • Lipidomics profiles from spastin mutant tissues to identify altered lipid species

  • Metabolomics analysis to detect changes in lipid synthesis and breakdown pathways

  • Integration with transcriptomics and proteomics to map regulatory networks

• Domain-Specific Manipulation:

  • Structure-function analysis of the hydrophobic motif (amino acids 57-86) critical for lipid droplet targeting

  • Generation of separation-of-function mutants that retain microtubule severing but lack lipid droplet binding

  • CRISPR-based editing of endogenous loci to introduce specific mutations

• Tissue and Cell-Type Specific Studies:

  • Comparative analysis across tissues with different responses to Spastin manipulation (neurons vs. muscles vs. fat bodies)

  • Single-cell approaches to detect cellular heterogeneity in responses

  • Creation of cell type-specific conditional models

• Therapeutic Targeting Strategies:

  • Screening for compounds that specifically modulate Spastin-lipid interactions

  • Testing lipid metabolism modulators in Spastin model systems

  • Dietary interventions to assess impact on Spastin-related phenotypes

These approaches will help elucidate whether altered lipid metabolism represents a potential therapeutic target in Spastin-related disorders and clarify the mechanistic links between Spastin's dual roles in cytoskeletal dynamics and lipid homeostasis.

How might new technological advances enhance our understanding of Spastin function in neuronal maintenance?

Emerging technological advances offer significant potential to deepen our understanding of Spastin function in neuronal maintenance:

• Advanced Microscopy Techniques:

  • Super-resolution microscopy to visualize microtubule network architecture at nanoscale resolution

  • Expansion microscopy to physically enlarge specimens for enhanced visualization of axonal structures

  • Light-sheet microscopy for long-term, non-phototoxic imaging of neuronal development and maintenance

• In Vivo Functional Imaging:

  • Genetically encoded indicators for microtubule dynamics in intact nervous systems

  • Calcium imaging to correlate structural changes with functional outcomes

  • Optogenetic control of Spastin activity with spatiotemporal precision

• Multi-organism Modeling Approaches:

  • Parallel testing in Drosophila, C. elegans, zebrafish, and mammalian models

  • Human iPSC-derived neurons from patients with SPAST mutations

  • Brain organoids to model complex 3D tissue environments

• CRISPR-Based Technologies:

  • Base editing for precise introduction of disease-associated mutations

  • CRISPRi/CRISPRa for temporal control of Spastin expression

  • CRISPR screening to identify genetic modifiers of Spastin function

• Computational and Systems Biology:

  • Machine learning for automated analysis of complex neuronal morphologies

  • Agent-based modeling of microtubule dynamics and severing events

  • Network analysis to identify convergent pathways in neurodegeneration

These technologies will enable researchers to address key questions about the temporal dynamics of axonal degeneration, the cell-autonomous versus non-cell-autonomous effects of Spastin dysfunction, and the mechanisms underlying the selective vulnerability of specific neuronal populations in Spastin-related disorders.

What interdisciplinary approaches might yield new insights into therapeutic strategies for Spastin-related disorders?

Interdisciplinary approaches offer promising avenues for developing therapeutic strategies for Spastin-related disorders:

• Metabolic Engineering and Lipid Biology:

  • Investigation of lipid metabolism modulators based on Spastin's role in lipid droplet regulation

  • Exploration of dietary interventions targeting specific lipid pathways

  • Development of lipid nanoparticles for targeted delivery of therapeutics

• Chemical Biology and Drug Discovery:

  • High-throughput screening for compounds that modulate Spastin-microtubule interactions

  • Structure-based drug design targeting non-catalytic functions of Spastin

  • Repurposing of FDA-approved drugs that impact cytoskeletal dynamics

• Bioengineering and Materials Science:

  • Biomaterial scaffolds to support axonal maintenance and regeneration

  • Nanoscale delivery systems for crossing the blood-brain barrier

  • Microfluidic devices for studying compartmentalized neuronal responses

• Computational Biology and Artificial Intelligence:

  • In silico modeling of Spastin structure and function

  • Machine learning algorithms to predict compound efficacy

  • Network analysis to identify alternative therapeutic targets

• Gene Therapy and Editing Technologies:

  • AAV-based delivery of wild-type Spastin to affected tissues

  • CRISPR approaches for correcting dominant mutations

  • Antisense oligonucleotides to modulate Spastin isoform expression

• Systems Biology and Multi-omics:

  • Integration of transcriptomics, proteomics, metabolomics, and lipidomics data

  • Identification of biomarkers for disease progression and therapeutic response

  • Elucidation of cellular pathways suitable for combinatorial targeting

This interdisciplinary approach acknowledges the complex nature of Spastin-related disorders and leverages diverse expertise to develop multi-faceted therapeutic strategies addressing both the primary genetic defects and their downstream consequences.