Phospho-DNM1 (Ser778) Antibody

Basic Research Questions

• What is the significance of Dynamin-1 phosphorylation at Serine 778 in neuronal research?

  Dynamin-1 (DNM1) is a 95 kDa GTPase protein that plays a crucial role in membrane trafficking processes, particularly in endocytosis and synaptic vesicle recycling. The phosphorylation at Serine 778 (Ser778) represents a critical regulatory mechanism that modulates dynamin function.

  Phosphorylation at this site is primarily driven by cyclin-dependent kinase 5 (CDK5) and has been implicated in various neuronal functions . Importantly, this post-translational modification regulates dynamin's involvement in synaptic vesicle recycling and other endocytic processes.

  Research suggests that the phosphorylation state of DNM1 at Ser778 can serve as a molecular switch that affects membrane trafficking dynamics. When studying neuronal function, monitoring this phosphorylation provides insights into activity-dependent changes in endocytic machinery and synaptic plasticity .

• What applications are most effective for Phospho-DNM1 (Ser778) antibody use in neural research?

  Phospho-DNM1 (Ser778) antibodies have been optimized for several key applications:

  Application	Typical Dilution	Key Considerations
  Western Blot	1:500-1:3000	Detects ~95 kDa band in neural lysates
  ELISA	1:20000	High sensitivity for quantitative analysis
  Cell-Based ELISA	As directed	Allows in situ detection in cultured cells

  Western blotting is particularly effective for detecting endogenous levels of phosphorylated dynamin in tissue lysates, especially from stimulated hippocampal neurons or brain synaptosomes . This method allows visualization of specific immunolabeling of the ~95 kDa band corresponding to phosphorylated dynamin.

  Cell-based ELISA provides a higher-throughput alternative for measuring relative changes in phosphorylation across multiple experimental conditions, enabling researchers to assess the effects of various treatments or activators on DNM1 phosphorylation .

• How is the specificity of Phospho-DNM1 (Ser778) antibody validated for research applications?

  The specificity of Phospho-DNM1 (Ser778) antibodies is validated through multiple complementary approaches:

  • Phosphatase treatment: Immunolabeling is completely eliminated when blots are treated with lambda phosphatase (1,200 units for 30 min), confirming phospho-specificity .

  • Blocking peptides: The antibody labeling can be blocked by the Ser778 phosphopeptide used as antigen, while the corresponding dephosphopeptide does not block immunolabeling .

  • In vitro phosphorylation: The antibody specifically labels the purified protein phosphorylated in vitro by CDK5 but not by other kinases like PKC .

  • Cross-reactivity testing: The antibody does not cross-react with other purified substrates of CDK5 (e.g., amphiphysin and synapsin) .

  These validation methods collectively ensure that the signal detected is specifically from phosphorylated Ser778 on dynamin 1, rather than from other phosphorylated proteins or unphosphorylated dynamin.

Experimental Design Questions

• What are the optimal conditions for using Phospho-DNM1 (Ser778) antibody in Western blot?

  For optimal Western blot results with Phospho-DNM1 (Ser778) antibody, consider the following protocol parameters:

  • Sample preparation: Use freshly prepared lysates from neural tissues, especially rat hippocampal lysates stimulated with forskolin to increase phosphorylation . For brain tissue, synaptosomal preparations (10 μg) provide enriched signal .

  • Gel electrophoresis: Use standard SDS-PAGE (typically 7.5-10% gels) to effectively separate proteins around the 95 kDa range.

  • Transfer conditions: Transfer to PVDF or nitrocellulose membranes using standard protocols.

  • Blocking: Use 5% BSA in TBST rather than milk, as milk contains phosphatases that may reduce signal.

  • Primary antibody dilution: 1:500 to 1:2000 in blocking buffer, incubated overnight at 4°C .

  • Detection method: HRP-conjugated secondary antibodies with enhanced chemiluminescence provide excellent sensitivity.

  The key to successful detection is preserving the phosphorylation status during sample preparation by including phosphatase inhibitors in all buffers and keeping samples cold throughout processing .

• How can I design experiments to investigate the relationship between dynamin phosphorylation and synaptic function?

  To investigate this relationship, a multi-faceted experimental approach is recommended:

  1. Stimulation paradigms: Compare dynamin phosphorylation at Ser778 under different neuronal activation protocols. Forskolin stimulation has been shown to enhance Ser778 phosphorylation . Design experiments with varying stimulation durations and intensities.

  2. Pharmacological manipulation: Use CDK5 inhibitors to block phosphorylation or phosphatase inhibitors to maintain phosphorylation, then assess effects on endocytosis rates using FM dyes or pHluorin-tagged synaptic vesicle proteins.

  3. Correlation with function: Perform parallel electrophysiology and phosphorylation measurements to correlate changes in synaptic efficacy with dynamin phosphorylation levels.

  4. Mutation studies: Compare wild-type dynamin with phospho-mimetic (S778D) and phospho-deficient (S778A) mutants in rescue experiments to directly test the functional significance of this modification.

  5. Temporal dynamics: Design time-course experiments following stimulation to track the relationship between phosphorylation state and recovery of synaptic function .

  This comprehensive approach will provide insights into how phosphorylation at Ser778 regulates dynamin function in the context of synaptic vesicle cycling.

• What controls should be included when using Phospho-DNM1 (Ser778) antibody in experimental designs?

  Rigorous controls are essential for interpreting results with Phospho-DNM1 (Ser778) antibody:

  • Phosphatase treatment control: Treat duplicate samples with lambda phosphatase to demonstrate phospho-specificity. This should eliminate signal from phosphorylated dynamin .

  • Total dynamin control: Use an antibody against total DNM1 to normalize phosphorylation levels to total protein expression. This is particularly important when comparing different experimental conditions .

  • Blocking peptide control: Include a lane where the antibody has been pre-incubated with the phosphopeptide immunogen to confirm specific binding .

  • Positive control: Include forskolin-stimulated neural samples known to have high levels of phosphorylated Ser778 .

  • Loading control: Use GAPDH (for whole-cell lysates) or appropriate synaptic markers (for synaptosomal preparations) to ensure equal loading .

  • Negative controls: Include non-neural tissues where DNM1 expression is low to confirm specificity.

  For cell-based ELISA applications, additional normalization methods should be employed, including Crystal Violet whole-cell staining to adjust for cell density differences across wells .

Data Interpretation Questions

• How can researchers distinguish between specific and non-specific bands when using Phospho-DNM1 (Ser778) antibody?

  Distinguishing specific from non-specific bands requires systematic analysis:

  1. Molecular weight verification: The specific band for phosphorylated DNM1 should appear at approximately 95 kDa .

  2. Phosphatase treatment: The specific band should disappear or be dramatically reduced after lambda phosphatase treatment, while non-specific bands will remain .

  3. Peptide competition: Pre-incubation of the antibody with the phosphopeptide immunogen should eliminate the specific band but not affect non-specific bands .

  4. Comparison with total DNM1: Running parallel blots with antibodies against total DNM1 can help confirm the identity of the band.

  5. Tissue specificity: The specific band should be strongest in neural tissues where DNM1 is predominantly expressed.

  6. Stimulation response: The intensity of the specific band should increase in response to treatments that activate CDK5 (like forskolin stimulation) .

  If multiple bands persist despite these controls, consider further optimization of blocking conditions or antibody dilution to improve specificity.

• What explains the variability in phospho-dynamin signal across different neural tissue preparations?

  Several factors contribute to variability in phospho-dynamin signal:

  1. Phosphorylation dynamics: Dynamin phosphorylation is highly dynamic and sensitive to neural activity. Even slight variations in tissue handling can affect the phosphorylation state .

  2. Regional differences: Different brain regions show varying levels of baseline dynamin phosphorylation, with hippocampal and cortical tissues typically showing higher levels than cerebellum .

  3. Developmental stage: DNM1 expression and phosphorylation patterns change during development, with important implications for studies comparing samples from different age groups .

  4. Sample preparation: The speed of tissue processing and inclusion of phosphatase inhibitors significantly impacts phosphorylation preservation.

  5. Subcellular fractionation: Synaptosomal preparations often show enriched and more consistent phospho-dynamin signal compared to whole homogenates .

  To minimize variability, standardize all aspects of tissue collection and processing, including rapid dissection, immediate freezing or homogenization in phosphatase inhibitor-containing buffers, and consistent subcellular fractionation protocols.

• How do I accurately quantify relative changes in dynamin phosphorylation at Ser778?

  Accurate quantification requires rigorous methodology:

  1. Normalization strategy: Always normalize phospho-DNM1 signal to total DNM1 levels to account for expression variations. For cell-based assays, additional normalization to GAPDH or cell number (via Crystal Violet staining) is recommended .

  2. Linear detection range: Ensure signals fall within the linear range of detection for your imaging system. Run a dilution series to confirm linearity.

  3. Technical replicates: Include at least three technical replicates per biological sample.

  4. Standardized exposure: For Western blots, use standardized exposure times or detection settings across experiments.

  5. Densitometry software: Use specialized software (ImageJ, Image Studio, etc.) for quantification, employing consistent region of interest selection methods.

  6. Statistical analysis: Apply appropriate statistical tests based on your experimental design, considering both biological and technical variability.

  For higher-throughput quantification, consider using cell-based ELISA methods that offer multiple normalization options and can accommodate more experimental conditions simultaneously .

Advanced Research Questions

• What is the relationship between CDK5 activity and DNM1 phosphorylation at Ser778 in neuronal function?

  The relationship between CDK5 and DNM1 phosphorylation reveals a complex regulatory mechanism:

  CDK5 directly phosphorylates DNM1 at serine residues 774 and 778 in neural cells . This phosphorylation plays a critical role in regulating dynamin's function during endocytosis, particularly in the context of synaptic vesicle recycling.

  Recent research has demonstrated that:

  • CDK5-mediated phosphorylation of DNM1 at Ser778 inhibits its GTPase activity, which temporarily prevents dynamin from participating in endocytosis during periods of high synaptic activity .

  • The phosphorylation state changes dynamically during synaptic activity cycles, with dephosphorylation by calcineurin (protein phosphatase 2B) activating dynamin for endocytosis following stimulation.

  • In studies examining spermatogenesis, strong co-localization of CDK1 and DNM2 was observed in the cytoplasm of spermatogonia and primary spermatocytes, with proximity ligation assay showing close association (<40 nm) . While this specifically examined DNM2, it suggests conserved kinase interactions across dynamin isoforms.

  • No significant interaction was observed between DNM2 and other potential phosphorylating kinases including CDK2, CDK5, or GSK3 in testicular cells , highlighting tissue-specific regulation of dynamin phosphorylation.

  This complex regulation suggests that CDK5-mediated phosphorylation of DNM1 at Ser778 acts as a molecular switch that coordinates endocytic capacity with neuronal activity levels.

• How does phosphorylation at Ser778 compare with other phosphorylation sites on dynamin 1?

  Dynamin 1 contains multiple phosphorylation sites that work in concert to regulate its function:

  Phosphorylation Site	Primary Kinase	Functional Impact	Temporal Dynamics
  Ser778	CDK5	Inhibits GTPase activity	Constitutively phosphorylated at rest
  Ser774	CDK5/GSK3	Inhibits self-assembly	Phosphorylated at rest
  Thr780	GSK3	Coordinates with S774	Secondary phosphorylation
  Ser857	PKC	Enhances lipid binding	Activity-dependent

  Ser778 phosphorylation is particularly notable because:

  1. It is primarily mediated by CDK5 rather than by a broader range of kinases .

  2. Its phosphorylation state is highly regulated during neuronal activity cycles.

  3. It works in concert with Ser774 phosphorylation, with evidence suggesting a sequential phosphorylation mechanism.

  4. Unlike some other sites, Ser778 phosphorylation directly affects dynamin's GTPase activity rather than just its assembly properties.

  5. Antibodies specific for phospho-Ser778 show less cross-reactivity with other phosphorylation sites compared to antibodies against other phospho-sites .

  Understanding the interplay between these phosphorylation sites provides insights into the complex regulation of dynamin function during endocytosis and other membrane trafficking events.

• What methods can be used to study the temporal dynamics of dynamin phosphorylation in live neurons?

  Investigating temporal dynamics of dynamin phosphorylation requires sophisticated approaches:

  1. Time-resolved immunocytochemistry: Fix neurons at defined time points after stimulation and stain with phospho-specific antibodies. This provides snapshots of phosphorylation states across the population .

  2. Phosphorylation-dependent FRET sensors: Design FRET-based reporters where the dynamin molecule is flanked by fluorescent proteins that undergo FRET changes upon phosphorylation-induced conformational changes.

  3. Phospho-mimetic and phospho-deficient mutants: Express dynamin variants with S778D (mimicking constitutive phosphorylation) or S778A (preventing phosphorylation) mutations fused to fluorescent tags to track their different dynamics during synaptic activity.

  4. Correlative electrophysiology and immunocytochemistry: Combine patch-clamp recording with post-hoc immunostaining for phospho-DNM1 to correlate functional states with phosphorylation levels.

  5. Optogenetic manipulation of CDK5 activity: Use light-activatable CDK5 constructs to precisely control the timing of phosphorylation events while monitoring functional outcomes.

  6. Microfluidic fast-perfusion systems: Rapidly apply and wash out kinase or phosphatase inhibitors during live imaging to track dynamic changes in dynamin localization and function.

  These approaches, used in combination, can provide unprecedented insights into the temporal regulation of dynamin phosphorylation during various aspects of neuronal function.

Methodology Questions

• How do cell-based ELISA and Western blot methods compare for measuring phospho-DNM1 (Ser778)?

  Both methods offer distinct advantages for phospho-DNM1 (Ser778) detection:

  Parameter	Western Blot	Cell-Based ELISA
  Sensitivity	Moderate to high	High
  Specificity	High (size verification)	Moderate (relies on antibody specificity)
  Throughput	Low to moderate	High
  Sample requirement	Cell/tissue lysates	Intact adherent cells
  Detection of multiple proteins	Possible on same membrane	Requires separate wells
  Quantification	Semi-quantitative	More precisely quantitative
  Time required	1-2 days	Several hours
  Technical complexity	Higher	Lower

  Cell-based ELISA offers significant advantages for screening multiple conditions, as it:

  1. Allows detection of phospho-DNM1 directly in cultured cells without lysate preparation .

  2. Provides multiple normalization options, including GAPDH, total DNM1, and cell density via Crystal Violet staining .

  3. Enables high-throughput screening of compounds or conditions affecting phosphorylation.

  Western blotting remains advantageous for:

  1. Confirming antibody specificity via molecular weight verification.

  2. Detecting phosphorylation in complex tissue samples like brain regions.

  3. Examining multiple proteins or phosphorylation sites on a single membrane.

  For comprehensive studies, using both methods complementarily provides the most robust data.

• What are the critical considerations for using Phospho-DNM1 (Ser778) antibody across different model organisms?

  When using Phospho-DNM1 (Ser778) antibody across species, consider these factors:

  1. Sequence conservation: The region surrounding Ser778 is highly conserved across mammals. Most commercial antibodies show reactivity with human, mouse, and rat samples . The exact epitope typically includes the phosphorylated sequence T-S-SP-P-T .

  2. Antibody validation in target species: Even with sequence conservation, empirical validation in each species is essential. Most commercial antibodies have been directly tested in rat tissue, with predicted reactivity in human, mouse, bovine, and canine samples based on sequence homology .

  3. Isoform specificity: Ensure the antibody specifically recognizes DNM1 and not other dynamin isoforms (DNM2, DNM3) that may have similar sequences but different functions across tissues .

  4. Dilution optimization: Optimal working dilutions may vary between species; for example, antibodies typically used at 1:500-1:2000 for rat samples may require adjustment for other species .

  5. Tissue expression patterns: DNM1 is predominantly expressed in neural tissues, so non-neural tissues may have lower signal regardless of antibody cross-reactivity.

  For evolutionary studies or when working with non-mammalian models, consider having custom antibodies generated against the specific sequence from your organism of interest.

• What advanced imaging techniques can be combined with Phospho-DNM1 (Ser778) antibody for spatial analysis?

  Several advanced imaging approaches can enhance spatial analysis of phospho-DNM1:

  1. Super-resolution microscopy: Techniques like STORM, PALM, or STED can resolve the localization of phosphorylated dynamin at the nanoscale level, revealing precise positioning at endocytic sites. This overcomes the diffraction limit of conventional microscopy.

  2. Proximity Ligation Assay (PLA): This technique can detect interactions between phospho-DNM1 and potential binding partners or kinases (like CDK5) when they are within 40 nm of each other, as demonstrated in studies examining dynamin interactions with CDK1 .

  3. Correlative Light and Electron Microscopy (CLEM): Combine immunofluorescence for phospho-DNM1 with electron microscopy to correlate phosphorylation state with ultrastructural features of endocytic intermediates.

  4. Expansion microscopy: This technique physically expands samples to achieve super-resolution imaging with standard confocal microscopes, allowing visualization of phospho-dynamin localization relative to synaptic structures.

  5. Live-cell imaging with phospho-sensors: Though challenging to develop, phosphorylation-sensitive fluorescent reporters could potentially allow real-time visualization of dynamin phosphorylation state changes.

  6. Single-molecule tracking: Apply this to study the mobility and clustering of individual dynamin molecules in relation to their phosphorylation state.

  These advanced techniques, particularly PLA for protein interaction studies and super-resolution approaches for precise localization, can reveal how phosphorylation regulates dynamin's spatial organization during endocytosis.