PFN1 Human

What is the molecular function of human Profilin-1 in cytoskeletal regulation?

Profilin-1 functions as a multifaceted cytoskeletal regulator through several distinct mechanisms. At the molecular level, PFN1 binds to G-actin monomers and catalyzes nucleotide exchange (ATP for ADP), preparing actin for incorporation into growing filaments . PFN1 also stimulates formin-mediated actin filament assembly, while simultaneously inhibiting Arp2/3-dependent nucleation, thereby serving as a gatekeeper allocating G-actin to specific assembly pathways . Additionally, PFN1 binds directly to tubulin dimers with a dissociation constant (kD) of 1.89 μM, enabling it to influence microtubule dynamics . This dual functionality makes PFN1 a potential coordinator between actin and microtubule networks, particularly in processes requiring synchronized cytoskeletal remodeling.

How can researchers effectively visualize PFN1 interactions in living cells?

Visualizing PFN1 in live cells has historically been challenging because conventional fusion and direct labeling strategies typically compromise PFN1 function. Recent advances have produced two genetically encoded tagged versions that maintain full functionality: Halo-PFN1 and mApple-PFN1 (mAp-PEN1) . Both versions successfully:

• Bind to phosphoinositide lipids

• Catalyze nucleotide exchange on actin monomers

• Stimulate formin-mediated actin filament assembly

• Bind tubulin dimers with normal affinity

• Restore morphological and cytoskeletal functions in PFN1-deficient cells

For optimal visualization, researchers can use titrations of self-labeling Halo-ligands, allowing precise control over labeling density . This approach, combined with function-disrupting point-mutants (Y6D and R88E) as controls, has successfully revealed PFN1 bound to microtubules in live cells .

What protein partners does PFN1 interact with to regulate cytoskeletal dynamics?

PFN1 orchestrates cytoskeletal regulation through interactions with numerous protein partners, each contributing to specialized aspects of actin and microtubule organization. Key interaction partners include:

• Formins: PFN1 delivers ATP-actin monomers to formins (such as diaphanous) at the barbed end of growing filaments, accelerating polymerization rates

• WASP family proteins: PFN1 interacts with neural Wiskott–Aldrich syndrome protein (N-WASP/WASL), influencing Arp2/3-dependent actin nucleation

• Ena/VASP family: PFN1 binds to mammalian enabled (Mena)/VASP family and enabled/VASP-like (Evl) proteins, which regulate actin dynamics at leading edges of migrating cells

• Adaptor proteins: PFN1 interacts with paladin, Rap1 GTP-interacting adaptor molecule (RIAM/APBB1IP), and lamellipodin (RAPH1)

These interactions occur primarily through binding of PFN1 to poly-proline motifs in these proteins, with conserved residues at positions 30 and 32 serving as key poly-proline binding sites .

What controls are essential when studying PFN1 mutants in experimental settings?

When studying PFN1 mutants, particularly disease-associated variants, several critical controls must be included to ensure reliable and interpretable results:

These controls help distinguish between specific functional deficits and general protein instability, providing more precise insights into mechanisms of PFN1 dysfunction in disease states .

How should researchers approach studying the dual role of PFN1 in actin and microtubule dynamics?

Studying PFN1's dual functionality requires integrated approaches that capture its simultaneous effects on both cytoskeletal systems. A methodological framework includes:

• Live-cell imaging: Utilize the fluorescently tagged PFN1 constructs (Halo-PFN1 or mApple-PFN1) that maintain full functionality to visualize PFN1's dynamic association with both actin and microtubule structures in real-time

• Domain-specific mutations: Employ targeted mutations that selectively disrupt interaction with either actin or microtubules:

  • Y6D and R88E specifically disrupt microtubule binding

  • G118V affects both actin and microtubule interactions

• Quantitative analysis: Measure:

  • Rates of actin polymerization in formin-mediated versus Arp2/3-dependent pathways

  • Microtubule growth and stability parameters

  • Co-localization coefficients between PFN1 and both cytoskeletal elements

• Biochemical validation: Confirm direct interactions using purified components to determine binding affinities and kinetic parameters for both actin and tubulin interactions

This multifaceted approach can reveal how PFN1 coordinates between the two major cytoskeletal systems and how disease-associated mutations might disrupt this coordination.

How do ALS-associated PFN1 mutations affect protein structure and function?

ALS-associated PFN1 mutations disrupt protein function through various mechanisms, with effects varying by specific mutation. Research findings indicate:

• Cytoskeletal Association Defects:

  • G118V variant fails to associate with both actin filaments and microtubules

  • Novel c.92T > G (p.Val31Gly) variant has been linked to ALS18 in a three-generation family with four affected individuals

• Protein Stability Effects:

  • Different mutations produce varying degrees of protein destabilization:

    • c.341T > C (p.Met114Thr) and c.353G > T (p.Gly118Val) partially destabilize PFN1

    • c.211T > G (p.Cys71Gly) causes severe destabilization at the microtubule level with increased accumulation of insoluble fractions

• Clinical Correlations:

  • The p.Val31Gly variant family showed a mean disease onset age of 59.75 (±10.11 SD) years

  • Significant intergenerational differences were observed: onset was 22.33 (±3.4 SD) years earlier in the third generation (male) compared to the first two generations (females)

  • Disease progression was relatively slow at 4 (±1.87 SD) years

• Molecular Mechanisms:

  • ALS-linked PFN1 variants exhibit both loss and gain of functions in formin-induced actin polymerization

  • Variants disturb microtubule dynamics and/or coordination between actin and microtubule filaments in motoneurons

Computational modeling using AlphaFold2 has been employed to simulate the structural impact of mutations, though the p.Val31Gly variant did not show obvious structural alterations despite its clinical effects .

What are the emerging links between PFN1 dysfunction and cardiovascular diseases?

PFN1 plays crucial roles in cardiovascular health, with dysregulation contributing to multiple cardiovascular pathologies. Research indicates that altered PFN1 expression and function are associated with atherosclerosis, hypertension, and diabetes . The actin cytoskeleton, regulated by PFN1, is fundamentally important for vascular cell functions including migration, proliferation, and response to mechanical stress.

Key mechanisms linking PFN1 to cardiovascular diseases include:

• Endothelial cell function: PFN1 regulates endothelial cell migration and angiogenesis, processes critical for vascular repair and remodeling

• Vascular smooth muscle cells (VSMCs): PFN1 influences VSMC proliferation and migration, which are central to vascular remodeling in hypertension and atherosclerosis

• Inflammatory responses: The cytoskeletal remodeling mediated by PFN1 impacts immune cell recruitment and function in vascular inflammation

• Cellular stress responses: PFN1 is involved in cellular responses to oxidative stress and mechanical forces, both of which are altered in cardiovascular diseases

Research approaches to investigate these connections include tissue-specific PFN1 knockout models, analysis of PFN1 expression in patient samples, and mechanistic studies of how PFN1 influences signal transduction pathways relevant to cardiovascular pathophysiology.

How might therapeutic strategies targeting PFN1 be developed for ALS and other diseases?

Developing therapeutic strategies targeting PFN1 requires understanding its mechanistic roles in disease and identifying approaches that can restore normal function. Potential therapeutic approaches include:

• Small molecule stabilizers: Developing compounds that bind to and stabilize disease-associated PFN1 mutants, preventing misfolding and aggregation. This approach would be particularly relevant for variants like c.211T > G (p.Cys71Gly) that cause severe protein destabilization

• Cytoskeletal modulators: Identifying molecules that can restore the balance between actin and microtubule dynamics disrupted by PFN1 mutations. This might involve compounds that selectively enhance or inhibit specific PFN1 interactions

• Gene therapy approaches: Delivering wild-type PFN1 to affected tissues to compensate for mutant protein dysfunction. The development of the fully functional tagged PFN1 constructs (Halo-PFN1 and mApple-PFN1) provides important tools for testing such approaches

• Targeting downstream effectors: Identifying and modulating the downstream consequences of PFN1 dysfunction, such as alterations in specific actin assembly pathways or microtubule stability

• Personalized approaches: Tailoring treatments based on specific mutations, as different PFN1 variants affect different aspects of protein function and stability. For example, the approaches for addressing G118V (which affects both actin and microtubule binding) would differ from those for variants that primarily affect protein stability

Research to develop these approaches will benefit from the tagged PFN1 constructs that allow visualization of protein dynamics in living cells and from a deeper understanding of how different mutations affect specific aspects of PFN1 function.

What experimental approaches can resolve conflicting data about PFN1's role in different cellular contexts?

Resolving conflicting data about PFN1's functions requires systematic approaches that account for context-dependent effects. Recommended methodologies include:

• Controlled expression systems: Develop inducible expression systems that allow precise control over PFN1 levels to determine whether contradictory effects are dose-dependent

• Cell type-specific analyses: Examine PFN1 functions across multiple cell types using identical methodologies to identify context-dependent effects. This is particularly important given the diverse roles of PFN1 in neuronal, cardiovascular, and other cell types

• Domain-specific mutations: Utilize mutations that selectively disrupt specific PFN1 functions (actin binding, microtubule binding, phosphoinositide interaction) to isolate which activities are responsible for specific cellular effects

• Standardized activity assays: Develop standardized in vitro assays for measuring specific PFN1 activities that can be consistently applied across different studies and laboratories

• Integrated multi-omics approaches: Combine proteomics, transcriptomics, and functional studies to create comprehensive models of PFN1's role in different cellular networks and pathways

• Temporal analysis: Investigate PFN1 function at different time points to determine whether apparently contradictory functions may be temporally separated during cellular processes

These approaches can help reconcile seemingly contradictory findings, such as PFN1's reported ability to both promote membrane protrusions and inhibit Arp2/3-dependent nucleation of actin filaments .