Recombinant Mouse Disheveled-associated activator of morphogenesis 2 (Daam2), partial

What is Recombinant Mouse Disheveled-associated activator of morphogenesis 2 (Daam2) and how is it produced for research?

Daam2 (Dishevelled-associated activator of morphogenesis 2) is a formin family member that plays a crucial role in cellular morphogenesis by mediating actin assembly and cytoskeletal remodeling . For research purposes, recombinant mouse Daam2 can be produced by subcloning PCR products from mouse full-length cDNA (such as GeneScript, OMu20712) . The production typically involves expression vector systems like pRK5-N-Myc, pCDNA6.2-N-GFP, or pSirenRetroQ . Researchers commonly use site-directed mutagenesis to introduce specific mutations that mimic those found in disease states or to create truncation constructs for functional domain analysis .

What fundamental cellular processes does Daam2 participate in?

Daam2 is fundamentally involved in:

• Actin cytoskeleton regulation and remodeling, essential for cellular morphogenesis

• Enhancement of canonical Wnt signaling during embryonic spinal cord patterning

• Regulation of oligodendrocyte (OL) lineage progression and differentiation

• Filopodia formation in cells, which can be assessed through specialized filopodia assays

• Podocyte function and integrity, with implications for kidney filtration barriers

The regulatory role of Daam2 in these processes is stage-specific, with evidence suggesting its inhibitory effect on early OL differentiation but potential promotive role during maturation and myelination stages .

How do researchers effectively design experiments to study Daam2 function?

When designing experiments to study Daam2 function, researchers should consider the following methodological approaches:

• Genetic manipulation techniques:

  • Design shRNA for Daam2 knockdown studies in relevant cell types

  • Create cDNA constructs reflecting wild-type Daam2 or specific variants for rescue experiments

  • Develop shRNA-resistant human cDNA clones to validate specificity of knockdown effects

• Functional assays:

  • Podocyte migration rate (PMR) assays to evaluate cellular motility

  • Filopodia formation assessments using videomicroscopy systems (e.g., IncuCyte) with GFP-tagged Daam2 constructs

  • Actin remodeling assessments using in vitro bulk and TIRF microscopy

• Protein interaction studies:

  • Immunoprecipitation followed by mass spectrometry (IP-MS) to identify Daam2-interacting proteins

  • Co-immunoprecipitation assays to confirm specific interactions, such as with INF2

  • Phosphorylation state analysis using phospho-null (e.g., S704A/T705A) or phospho-mimetic (e.g., S704E/T705E) mutants

What is the relationship between Daam2 phosphorylation and its functional activity?

Daam2 phosphorylation represents a critical regulatory mechanism that modulates its functional activity. Mass spectrometric analysis has identified phosphorylation sites at residues S704 and T705 in the FH2 domain of mouse Daam2 . These sites are highly conserved across species, suggesting their evolutionary importance in Daam2 function regulation.

The functional significance of these phosphorylation events has been experimentally demonstrated through the introduction of phospho-null (S704A/T705A, "A-mutant") and phospho-mimetic (S704E/T705E, "E-mutant") mutations:

These findings demonstrate that phosphorylation of Daam2 at S704/T705 serves as a molecular switch that converts Daam2 from an inhibitor to a promoter of oligodendrocyte differentiation . The kinase CK2α has been identified as responsible for this phosphorylation, with compelling evidence showing that CK2α promotes OL differentiation specifically through Daam2 phosphorylation .

How do Daam2 variants affect actin cytoskeleton dynamics and cellular function?

Daam2 variants significantly impact actin cytoskeleton dynamics, with profound consequences for cellular function. Whole-exome sequencing has identified bi-allelic variants in DAAM2 in individuals with steroid-resistant nephrotic syndrome, linking Daam2 dysfunction to kidney disease .

Functional analysis of these variants reveals:

• Impaired actin remodeling: Wild-type DAAM2 cDNA, but not cDNA representing disease-associated missense variants, can rescue the reduced podocyte migration rate in DAAM2-knockdown cells .

• Compromised filopodia formation: Disease-associated variants fail to restore filopodia formation in DAAM2-knockdown podocytes, unlike wild-type DAAM2 .

• Altered protein interactions: DAAM2 co-localizes and co-immunoprecipitates with INF2, another formin associated with nephrotic syndrome, suggesting potential functional interactions between these actin regulators .

These findings establish a mechanistic link between Daam2 variants and cytoskeletal dysregulation, particularly in specialized cell types like podocytes that rely heavily on precise actin organization for their function.

What role does Daam2 play in oligodendrocyte differentiation and myelination?

Daam2 exhibits a complex, stage-dependent role in oligodendrocyte (OL) differentiation and myelination processes:

• Inhibitory role in early differentiation: Daam2 overexpression significantly inhibits OL differentiation, while loss of Daam2 promotes differentiation during development and after white matter injury . This is evidenced by changes in mature OL markers like MAG+ and MBP+ cells following Daam2 manipulation.

• Potential positive role in maturation: Loss of Daam2 leads to abnormal myelin formation that normalizes at later stages, suggesting a potential promotive role during maturation and myelination phases .

• Regulation through phosphorylation: The inhibitory effect of Daam2 on OL differentiation can be reversed through phosphorylation by CK2α at residues S704/T705 . The phospho-mimetic E-mutant (S704E/T705E) promotes rather than inhibits OL differentiation.

• Relevance to pathological conditions: Daam2 is upregulated in demyelinated lesions in conjunction with higher Wnt signaling in hypoxic-ischemic encephalopathy (HIE) and multiple sclerosis (MS) patients . This suggests Daam2 as a potential therapeutic target for demyelinating disorders.

The dual role of Daam2 in OL development raises important questions about whether Wnt signaling plays a negative role in early differentiation but a positive role during maturation and myelination, highlighting the complexity of Daam2 function in the oligodendrocyte lineage .

What are the optimal approaches for studying Daam2 phosphorylation in experimental settings?

Studying Daam2 phosphorylation requires a multi-faceted experimental approach:

• Identification of phosphorylation sites:

  • Immunoprecipitation followed by mass spectrometry (IP-MS) using cortical tissues

  • Comparison of identified sites with phosphoproteomics databases to confirm previously reported sites (e.g., S704 phosphorylation in mice, equivalent to S656 in humans)

• Functional analysis of phosphorylation:

  • Generation of phospho-null mutants (e.g., S704A/T705A) that cannot be phosphorylated

  • Creation of phospho-mimetic mutants (e.g., S704E/T705E) that simulate constitutive phosphorylation

  • Transfection of these constructs into primary oligodendrocyte precursor cells (OPCs) to assess differentiation effects

• Identification of responsible kinases:

  • Co-immunoprecipitation assays to identify kinases that interact with Daam2

  • In vitro kinase assays to confirm direct phosphorylation

  • Use of kinase inhibitors to validate the role of specific kinases (e.g., CK2α)

• In vivo validation:

  • Introduction of adeno-associated virus (AAV) vectors expressing kinases (e.g., AAV-CK2α) or controls into wild-type and mutant mouse brains

  • Assessment of OL differentiation through markers like CC1+/tdTomato+

  • Comparison between different genetic backgrounds (wild-type vs. phospho-mimetic mutants) to confirm pathway specificity

How can researchers effectively assess Daam2's impact on actin dynamics?

Researchers can employ several specialized techniques to assess Daam2's impact on actin dynamics:

• Podocyte migration rate (PMR) assays:

  • Knockdown Daam2 expression using shRNA

  • Rescue experiments with wild-type or variant Daam2 cDNA constructs

  • Quantitative measurement of migration rates to assess functional consequences of Daam2 manipulation

• Filopodia formation assays:

  • Transfection of cultured human podocytes with GFP-tagged Daam2 constructs (wild-type or harboring human variants)

  • Time-lapse imaging using systems like IncuCyte Videomicroscopy over 24 hours

  • Quantitative assessment of filopodia number, length, and dynamics

• Pharmacological interventions:

  • Use of formin-activating molecules like IMM-01 to restore filopodia formation in Daam2-knockdown cells

  • Dose-response studies to determine optimal concentrations for specific cellular contexts

• Advanced microscopy techniques:

  • In vitro bulk actin polymerization assays

  • Total internal reflection fluorescence (TIRF) microscopy to visualize actin dynamics at high resolution

  • Fluorescent labeling of actin and Daam2 to track co-localization and dynamic interactions

What disease models are most appropriate for studying Daam2 dysfunction?

Several disease models have proven valuable for studying Daam2 dysfunction:

• Demyelination models:

  • Lysolecithin-induced demyelination in mouse corpus callosum, which mimics multiple sclerosis (MS) pathology

  • Assessment of myelin repair following Daam2 manipulation through CK2α-induced phosphorylation

• Nephrotic syndrome (NS) models:

  • In vitro podocyte culture systems with Daam2 knockdown or expression of disease-associated variants

  • Functional assays measuring podocyte migration and filopodia formation

• Genetic models:

  • Transgenic mice expressing phospho-null (A-mutant) or phospho-mimetic (E-mutant) Daam2

  • Conditional knockout models to study tissue-specific Daam2 functions

• Human disease tissue:

  • Analysis of Daam2 expression in demyelinated lesions from MS patients

  • Examination of DAAM2 variants in individuals with steroid-resistant nephrotic syndrome

These models collectively enable researchers to investigate Daam2's roles in different pathological contexts and evaluate potential therapeutic strategies targeting Daam2 or its regulatory pathways.

How do DAAM2 variants contribute to nephrotic syndrome pathogenesis?

Bi-allelic variants in DAAM2 have been identified as a monogenic cause of nephrotic syndrome (NS), adding to the >60 genetic causes of this condition . The pathogenic mechanisms involve:

• Disruption of podocyte actin cytoskeleton: Wild-type DAAM2 cDNA, but not disease-associated variants, can rescue reduced podocyte migration rates in DAAM2-knockdown cells, indicating that the variants impair DAAM2-dependent actin remodeling .

• Impaired filopodia formation: Disease-associated variants fail to restore filopodia formation in knockdown podocytes, compromising podocyte morphology and function .

• Altered interactions with other actin regulators: DAAM2 co-localizes and co-immunoprecipitates with INF2, another formin associated with NS. This suggests a potential interaction network among formins in maintaining podocyte function, which may be disrupted by disease-causing variants .

• Connection to RhoA/Rac1/Cdc42 signaling: The discovery of DAAM2 variants in NS reinforces the central role of actin regulators RhoA/Rac1/Cdc42 and their effectors in podocyte biology and NS pathogenesis .

These findings establish DAAM2 as an important component of the actin regulatory machinery in podocytes and explain how its dysfunction contributes to the development of steroid-resistant nephrotic syndrome.

What therapeutic approaches might target Daam2 in demyelinating disorders?

The involvement of Daam2 in oligodendrocyte differentiation and myelination suggests several potential therapeutic approaches for demyelinating disorders:

• Modulation of Daam2 phosphorylation:

  • Enhancing CK2α activity or expression to promote Daam2 phosphorylation at S704/T705, which converts Daam2 from an inhibitor to a promoter of OL differentiation

  • Development of small molecules that mimic the effect of phosphorylation on Daam2 conformation and function

• Formin-activating molecules:

  • Compounds like IMM-01, which have been shown to restore filopodia formation in Daam2-compromised cells, might enhance remyelination in demyelinating disorders

  • Screening for additional molecules that specifically modulate Daam2 activity in oligodendrocytes

• Targeting Wnt signaling:

  • As Daam2 enhances canonical Wnt signaling, and higher Wnt tone is observed in demyelinated lesions in hypoxic-ischemic encephalopathy (HIE) and MS patients, Wnt pathway modulators might indirectly affect Daam2 function

  • Development of stage-specific interventions that account for the potentially dual role of Wnt signaling in OL differentiation versus maturation

• Gene therapy approaches:

  • AAV-mediated delivery of CK2α or phospho-mimetic Daam2 to demyelinated lesions, as demonstrated in the lysolecithin-induced demyelination model

  • CRISPR-based approaches to introduce phospho-mimetic mutations in endogenous Daam2

These therapeutic strategies would need to account for the stage-specific roles of Daam2 in OL development and the potential systemic effects of Daam2 modulation given its roles in multiple tissues and cell types.

What are the critical quality control parameters for recombinant Daam2 production?

Ensuring high-quality recombinant Daam2 for research applications requires rigorous quality control:

• Sequence verification:

  • Confirmation of the complete coding sequence through DNA sequencing

  • Verification that site-directed mutagenesis has correctly introduced desired mutations

  • Checking for unintended mutations that might arise during the cloning process

• Expression validation:

  • Western blotting to confirm protein expression at the expected molecular weight

  • Immunofluorescence to verify subcellular localization (cytoplasmic in podocytes and kidney sections)

  • Assessment of post-translational modifications, particularly phosphorylation at key sites (S704/T705 in mouse Daam2)

• Functional validation:

  • Verification that wild-type recombinant Daam2 suppresses OL differentiation as expected

  • Confirmation that phospho-null and phospho-mimetic mutants exhibit anticipated functional differences

  • Assessment of the protein's ability to influence actin dynamics in appropriate cellular contexts

• Stability and storage parameters:

  • Determination of optimal buffer conditions for maintaining protein stability

  • Establishment of appropriate storage temperatures and freeze-thaw tolerance

  • Assessment of activity retention over time under various storage conditions

How can contradictory findings about Daam2 function be reconciled in experimental design?

Research on Daam2 has yielded some apparently contradictory findings that require careful experimental design to reconcile:

• Stage-specific functions:

  • Loss of Daam2 promotes OL differentiation during development but leads to abnormal myelin formation that normalizes at later stages

  • Experimental designs should include multiple time points to capture the dynamic, stage-dependent effects of Daam2

• Context-dependent activities:

  • Daam2's effects may vary depending on cell type, developmental stage, and pathological context

  • Studies should explicitly define and control for these contextual factors

  • Parallel experiments in multiple systems can help identify consistent versus context-specific effects

• Functional modulation by post-translational modifications:

  • Phosphorylation converts Daam2 from an inhibitor to a promoter of OL differentiation

  • Experiments should assess the phosphorylation state of Daam2 and account for how this might influence functional outcomes

  • Use of phospho-specific antibodies or phospho-mimetic/null mutants can help distinguish between different functional states

• Integration of in vitro and in vivo findings:

  • In vitro studies may not fully recapitulate the complex environment in which Daam2 functions in vivo

  • Validation of key findings across multiple experimental systems (cell culture, organoids, animal models)

  • Careful consideration of species differences when translating between mouse and human studies

By addressing these considerations in experimental design, researchers can develop a more nuanced understanding of Daam2's multifaceted roles in development and disease.