Recombinant Pongo abelii Atlastin-1 (ATL1)

Basic Research Questions

• What is the molecular structure and function of Atlastin-1 in Pongo abelii?

  Pongo abelii (Sumatran orangutan) Atlastin-1 is a dynamin-like GTPase of approximately 64 kDa with 558 amino acids. The protein contains a GTPase domain, a middle domain, a membrane-associated wedge motif, and a C-terminal amphipathic helix. It functions primarily in endoplasmic reticulum (ER) morphology by mediating homotypic fusion of ER tubules to form the characteristic three-way junctions of ER networks . The amino acid sequence includes key functional regions that enable GTP binding and hydrolysis, with conserved domains similar to human Atlastin-1 .

• What are the optimal storage conditions for recombinant Pongo abelii ATL1?

  For optimal stability of recombinant Pongo abelii ATL1, store at -20°C in Tris-based buffer with 50% glycerol. For extended storage, -80°C is recommended. Avoid repeated freeze-thaw cycles, as this significantly decreases protein activity. Working aliquots can be stored at 4°C for up to one week. The stability is maintained in optimized buffers containing 50% glycerol, which prevents protein denaturation during freezing .

• How can I confirm the identity and integrity of recombinant Pongo abelii ATL1?

  Verification should involve multiple approaches:

  • Western blotting using specific antibodies (such as 12149-1-AP or D2E6 Rabbit mAb) that have cross-reactivity with Pongo abelii ATL1

  • Mass spectrometry to confirm molecular weight (approximately 64 kDa)

  • GTPase activity assay to verify functional integrity

  • Circular dichroism spectroscopy to assess secondary structure

  • Assessment of purity via SDS-PAGE (should show >95% purity)

  Research has demonstrated that antibodies raised against human ATL1 often cross-react with Pongo abelii ATL1 due to high sequence homology .

• What expression systems are suitable for producing recombinant Pongo abelii ATL1?

  Several expression systems have proven effective:

  Expression System	Yield	Advantages	Limitations
  E. coli	Moderate	Cost-effective, rapid	May lack post-translational modifications
  Insect cells	High	Proper folding, some PTMs	Higher cost, longer production time
  Mammalian cells (HEK293)	Moderate-High	Native-like PTMs, correct folding	Highest cost, complex protocols

  For structural studies requiring large quantities, E. coli systems with optimized codons have been successful. For functional studies where proper folding is critical, mammalian or insect cell expression is preferable .

Advanced Research Questions

• How do mutations in ATL1 affect its GTPase activity and what methodologies are best for studying these effects?

  Mutations in ATL1 can significantly impair its GTPase activity, as observed in hereditary spastic paraplegia (HSP) cases. The GTPase activity can be assessed using:

  • Radiometric assays measuring the release of inorganic phosphate from γ-32P-GTP

  • Colorimetric assays using malachite green to detect released phosphate

  • FRET-based real-time assays with fluorescently labeled GTP analogs

  Research on the F151S mutation revealed impaired nucleotide-hydrolysis while maintaining the ability to form homodimers with transition-state analogs . For Pongo abelii ATL1, comparative GTPase assays with human variants can be particularly informative for understanding conserved mechanisms. Analysis should include both steady-state kinetics (Km and kcat) and pre-steady-state measurements to capture the complete catalytic cycle .

• What is the role of Pongo abelii ATL1 in endosomal tubulation and lysosomal proteolysis, and how can this be experimentally investigated?

  Recent studies with human ATL1 have shown that beyond ER morphology regulation, atlastin-1 affects endosomal tubulation and lysosomal proteolytic capacity. Neurons lacking atlastin-1 exhibit longer endosomal tubules and reduced lysosomal proteolysis . For studying Pongo abelii ATL1's role:

  1. Use CRISPR-inhibition to generate neuronal cells lacking ATL1

  2. Quantify endosomal tubule length using fluorescent markers

  3. Assess lysosomal proteolytic capacity with DQ-BSA degradation assays

  4. Examine three-way ER junction formation with super-resolution microscopy

  Comparative studies between human and Pongo abelii ATL1 would help identify conserved mechanisms in endolysosomal function. The experimental approach should include rescue experiments with wild-type and mutant ATL1 to confirm specificity of observed phenotypes .

• How can I design experiments to study the interaction between Pongo abelii ATL1 and microtubules in dendritic morphogenesis?

  Studies in C. elegans have revealed that ATL1 affects microtubule stability in dendrites, with ATL1 mutants showing reduced microtubules in dendritic branches . To investigate this with Pongo abelii ATL1:

  1. Express fluorescently tagged α-tubulin and ATL1 to monitor co-localization in neuronal cultures

  2. Perform time-lapse imaging to track microtubule dynamics in dendrites

  3. Use CRISPR to generate ATL1-knockout neurons and assess microtubule stability

  4. Conduct rescue experiments with wild-type and GTPase-deficient ATL1 mutants

  5. Employ super-resolution microscopy to visualize ER-microtubule contacts

  This approach would help determine whether the ATL1-microtubule relationship is conserved across species and illuminate the molecular mechanisms by which ATL1 influences dendritic morphology .

• What experimental approaches can differentiate between the roles of ATL1, ATL2, and ATL3 in neuronal development when using recombinant Pongo abelii proteins?

  Studies in human neurons show a developmental switch where ATL1 becomes the predominant atlastin during neuronal differentiation . To investigate this with Pongo abelii atlastins:

  1. Quantify relative expression of ATL1, ATL2, and ATL3 during neuronal differentiation using qPCR and immunoblotting

  2. Generate specific knockdowns of each atlastin to assess their individual contributions

  3. Perform rescue experiments with species-specific atlastins to determine functional conservation

  4. Use domain-swapping experiments between atlastin paralogs to identify functionally critical regions

  A time-course analysis during neuronal differentiation would be particularly informative, as human studies show ATL1 becoming predominant by day 7 of differentiation . Comparative analysis between human and Pongo abelii atlastins could reveal evolutionary adaptations in neuronal development.

• How can I establish a reliable in vitro membrane fusion assay to study the function of recombinant Pongo abelii ATL1?

  In vitro membrane fusion assays are critical for understanding ATL1 function. A recommended protocol includes:

  1. Prepare proteoliposomes containing purified recombinant ATL1:

     • Use synthetic lipids mimicking ER composition (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine)

     • Incorporate ATL1 at physiologically relevant protein:lipid ratios (1:1000 to 1:2000)

  2. Label separate proteoliposome populations with:

     • Fluorescence donor (NBD-PE)

     • Fluorescence acceptor (Rhodamine-PE)

  3. Monitor fusion kinetics by measuring FRET efficiency changes upon mixing

  4. Compare GTPase activity (measured separately) with fusion efficiency to establish structure-function relationships

  This assay can also be used to compare wild-type and mutant versions of ATL1, providing insights into how disease-associated mutations affect membrane fusion capability .

• What methods can be used to investigate the differential effects of homozygous versus heterozygous ATL1 mutations using Pongo abelii ATL1 as a model?

  Investigating differential effects of homozygous versus heterozygous ATL1 mutations requires sophisticated approaches:

  1. Generate in vitro mixtures of wild-type and mutant recombinant proteins at different ratios to mimic heterozygous states

  2. Assess dominant-negative effects in COS-7 cells:

     • Express Pongo abelii ATL1 mutants in cells with endogenous atlastins

     • Quantify 3-way junction density in ER networks

     • Compare effects of P208L (loss-of-function) and K80A (dominant-negative) mutations

  3. For homozygous effects, use cells lacking endogenous atlastins and express only mutant versions

  Research on human ATL1 has shown that mutations like P208L (equivalent to P219L in C. elegans) represent loss-of-function rather than dominant-negative mutations, as they don't affect ER morphology when expressed in wild-type cells . Similar approaches with Pongo abelii ATL1 would provide comparative insights into evolutionary conservation of these mechanisms.

• How does the neuron-specific expression pattern of ATL1 differ between humans and Pongo abelii, and what methodologies are appropriate for studying these differences?

  Human ATL1 shows neuron-specific expression patterns, being particularly abundant in cortical neurons. To investigate potential differences in Pongo abelii:

  1. Perform comparative transcriptomics:

     • RNA-seq of matched tissue samples from both species

     • Single-cell RNA-seq to identify cell-type specific expression patterns

  2. Use immunohistochemistry with cross-reactive antibodies on tissue sections

  3. Develop iPSC models from both species and differentiate to neurons:

     • Track ATL1 expression during differentiation with qPCR and immunoblotting

     • Compare expression switches between ATL1, ATL2, and ATL3

  Studies in human neurons showed ATL1 becoming the predominant atlastin by day 7 of neuronal differentiation, with ATL2 and ATL3 expression reducing . Determining whether this developmental switch occurs similarly in Pongo abelii would provide insights into the evolution of neuronal ER organization.

• What experimental approaches can assess how disease-associated ATL1 mutations affect endosomal tubulation and lysosomal proteolysis in Pongo abelii models?

  Recent research has linked ATL1 mutations to altered endosomal tubulation and reduced lysosomal proteolytic capacity . To study these effects using Pongo abelii ATL1:

  1. Generate neuronal models expressing wild-type or mutant Pongo abelii ATL1:

     • Use CRISPR-inhibition for endogenous ATL1 depletion

     • Rescue with wild-type or disease-associated mutant variants

  2. Assess endosomal morphology:

     • Quantify endosomal tubule length using live-cell imaging

     • Measure tubule fission rates with fluorescent endosomal markers

  3. Evaluate lysosomal proteolytic function:

     • DQ-BSA degradation assays

     • Cathepsin activity measurements

     • Pulse-chase experiments with labeled proteins

  4. Investigate changes in ER-endosome contact sites using proximity ligation assays

  This comprehensive approach would reveal whether the link between ATL1 function and endolysosomal health is conserved across species and provide insight into the pathogenic mechanisms of HSP-associated mutations .

Data Tables and Research Findings

• Table: Key Structural and Functional Domains of Pongo abelii ATL1

  Domain	Position	Function	Key Residues
  GTPase domain	1-339	GTP binding and hydrolysis	K80 (GTP binding)
  Middle domain	340-450	Dimerization and membrane tethering	F151 (allosteric coupling)
  Membrane-associated wedge	451-520	Membrane insertion, curvature sensing	P208/P219 (HSP-associated site)
  Amphipathic helix	521-558	Membrane disorder induction, fusion	Hydrophobic residues

  This domain organization is critical for ATL1's function in homotypic ER membrane fusion, with mutations in key residues leading to hereditary spastic paraplegia and other neurological disorders .

• Table: Comparison of ATL1 Expression During Neuronal Differentiation

  Differentiation Stage	ATL1	ATL2	ATL3	Main Function
  iPSCs	<3% of ATL2	Predominant	~28% of ATL2	ER maintenance in stem cells
  Day 7 neurons	Predominant	~41% of ATL1	~7% of ATL1	Early neuronal ER organization
  Day 14 neurons	Predominant	~41% of ATL1	~7% of ATL1	Mature neuronal ER maintenance
  Day 28 neurons	Predominant	Further reduced	Further reduced	Long-term ER network stability

  This expression pattern switch, documented in human neurons, suggests a specialized role for ATL1 in neuronal ER morphology. Similar studies in Pongo abelii would determine whether this developmental regulation is evolutionarily conserved .

• Figure: Effect of ATL1 Deficiency on ER Network Formation and Endosomal Function

  Research findings demonstrate that ATL1 deficiency leads to:

  1. Reduced number of three-way junctions in ER networks

  2. Increased unbranched, parallel ER tubules

  3. Longer endosomal tubules (suggestive of defective tubule fission)

  4. Reduced lysosomal proteolytic capacity

  5. Altered neurite growth (longer developing axons in ATL1-depleted neurons)

  These phenotypes were observed in human neurons and C. elegans models, suggesting conserved functions that may extend to Pongo abelii ATL1 .