PFN4 Human

What is PFN4 and how does it differ structurally from other profilin family members?

PFN4 (Profilin-4) is a member of the profilin family of small actin-binding proteins. Unlike other profilins, PFN4 shows only 30% homology to other family members (PFN1-3) and notably lacks the canonical actin and poly-L-proline binding sites . This structural divergence suggests that PFN4 has evolved functions independent of the typical actin dynamics regulation associated with other profilins.

Methodologically, researchers can investigate these structural differences through:

• Sequence alignment and phylogenetic analysis

• Structural studies using X-ray crystallography or cryo-EM

• Domain-function relationship studies using recombinant protein variants

What is the expression pattern of PFN4 in human tissues?

PFN4 exhibits highly specific expression, being predominantly expressed in testes during spermiogenesis . Within testicular tissue, PFN4 localizes specifically to the acrosome-acroplaxome-manchette complex of developing sperm cells . This restricted expression pattern suggests a specialized role in sperm development and maturation.

For researchers studying PFN4 expression:

• qRT-PCR can quantify mRNA levels across different tissues and developmental stages

• Immunohistochemistry allows visualization of PFN4's spatial distribution

• Single-cell RNA sequencing can reveal expression patterns in specific cell populations

What methodologies are most effective for investigating PFN4's role in manchette formation?

The manchette is a transient microtubular structure crucial for sperm head shaping and protein transport during spermiogenesis. Research has demonstrated that PFN4 deficiency severely impairs manchette formation, resulting in abnormal sperm head morphology and flagellar defects .

Recommended methodological approaches include:

• Genetic modification models: CRISPR/Cas9-mediated gene editing to generate PFN4-deficient models, as demonstrated in mouse studies .

• Advanced microscopy:

  • Immunofluorescence with antibodies against manchette components (e.g., HOOK1, ARL3)

  • Transmission electron microscopy (TEM) for ultrastructural analysis

  • Super-resolution microscopy for detailed spatial organization

• Molecular interaction studies:

  • Co-immunoprecipitation to identify PFN4 binding partners

  • Proximity labeling techniques to map the PFN4 interactome in situ

  • Live-cell imaging with fluorescently tagged proteins to monitor dynamics

Current evidence suggests that while PFN4 deficiency does not affect perinuclear ring formation or initial HOOK1 localization, it severely impedes microtubular organization of the manchette, as shown by disrupted ARL3 staining patterns .

How does PFN4 deficiency affect signaling pathways during spermatogenesis?

Proteomic analysis of PFN4-deficient testes has revealed significant alterations in several key signaling pathways, with potential implications for understanding the molecular mechanisms underlying the observed phenotypes .

For researchers investigating these pathways:

• Western blotting can confirm changes in protein levels and phosphorylation states

• Pharmacological inhibitors of specific pathway components can establish causality

• Time-course experiments during spermatogenesis can determine sequential events

• Rescue experiments can test whether pathway modulation restores normal phenotypes

Evidence suggests these pathway alterations collectively result in inhibited autophagy, as indicated by increased LC3I/II and SQSTM1 protein levels, potentially explaining the observed defects in acrosome formation .

What is the relationship between PFN4 and acrosome biogenesis?

PFN4 plays a critical role in acrosome formation during spermiogenesis. PFN4-deficient mice exhibit severe defects in acrosome biogenesis, characterized by :

• Disrupted cis- and trans-Golgi networks

• Aberrant production of proacrosomal vesicles

• Impaired formation of the acrosomal vesicle

Methodological approaches to investigate this relationship include:

• Ultrastructural analysis: TEM examination at sequential stages of acrosome development

• Vesicular trafficking assays: Tracking the movement of Golgi-derived vesicles

• Golgi integrity assessment: Immunolabeling of Golgi markers in PFN4-deficient cells

• Autophagy flux measurement: Quantifying LC3 turnover and p62/SQSTM1 clearance

Current evidence suggests that PFN4 deficiency disrupts Golgi function and vesicular trafficking, potentially through alterations in the PI3K/AKT/mTOR signaling axis and consequent inhibition of autophagy, which collectively impair acrosome formation .

How can researchers differentiate between direct and indirect effects of PFN4 deficiency?

Distinguishing primary from secondary effects of PFN4 deficiency presents a significant methodological challenge. Researchers should consider:

• Temporal analysis: Establishing a timeline of cellular and molecular events following PFN4 depletion can help identify initial changes.

• Conditional knockout models: Using stage-specific or cell-type-specific PFN4 deletion to isolate direct effects.

• Rescue experiments: Testing whether reintroduction of PFN4 or modulation of downstream pathways can reverse specific phenotypes.

• In vitro reconstitution: Reconstructing minimal systems with purified components to test direct biochemical interactions.

• Comparative analysis: Examining phenotypic overlap with other genetic models affecting manchette formation or acrosome biogenesis.

Evidence suggests that while manchette formation defects appear to be a direct consequence of PFN4 deficiency, some acrosomal abnormalities may be secondary to altered PI3K/AKT signaling and autophagy inhibition .

What are the optimal methods for producing and utilizing recombinant PFN4 protein in research?

Recombinant PFN4 production is essential for biochemical and structural studies. Based on available data :

Human PFN4 can be produced in E. coli as a single, non-glycosylated polypeptide chain containing 149 amino acids (1-129 a.a.) with a molecular mass of 16.4 kDa. Typical production involves:

• Expression system optimization:

  • E. coli is suitable for basic structural studies

  • Mammalian or insect cell systems may be preferred if post-translational modifications are suspected

• Purification strategy:

  • N-terminal His-tag facilitates purification via standard chromatography methods

  • Size exclusion chromatography can ensure homogeneity

• Functional validation:

  • Circular dichroism to confirm proper folding

  • Limited proteolysis to assess structural integrity

  • Binding assays with potential interaction partners

• Application considerations:

  • In vitro reconstitution assays

  • Structural studies (X-ray crystallography, NMR, or cryo-EM)

  • Pull-down experiments to identify binding partners

Researchers should be aware that the absence of actin and poly-L-proline binding sites in PFN4 necessitates alternative approaches for functional characterization compared to other profilins .

What contradictions exist in current PFN4 research and how can they be resolved?

Current PFN4 research contains several unresolved questions and apparent contradictions that merit methodological investigation:

• Function despite lacking canonical binding sites:
  While PFN4 lacks the typical actin and poly-L-proline binding sites of other profilins, it still influences cytoskeletal structures. This apparent contradiction requires:

  • Identification of alternative binding partners

  • Characterization of novel functional domains

  • Investigation of potential indirect effects on cytoskeletal dynamics

• Relationship between autophagy inhibition and manchette formation:
  The causal relationship between observed autophagy inhibition and manchette defects remains unclear. Resolving this requires:

  • Time-course studies to establish sequence of events

  • Selective autophagy modulation without affecting PFN4

  • Mechanistic studies of autophagy's role in manchette formation

• Variability in fertility outcomes:
  While homozygous PFN4-deficient males are completely infertile, heterozygotes retain normal fertility despite reduced PFN4 mRNA levels . This suggests:

  • Potential threshold effects requiring quantitative analysis

  • Compensatory mechanisms that merit investigation

  • Dose-dependent studies of PFN4 function

What is the potential clinical significance of PFN4 in human male infertility disorders?

Based on mouse model studies, PFN4 deficiency results in male infertility characterized by severe manchette formation defects, abnormal sperm head morphology, and impaired acrosome biogenesis . These findings suggest PFN4 may have clinical relevance in cases of:

• Teratozoospermia: Abnormal sperm morphology, particularly affecting head shape

• Asthenozoospermia: Reduced sperm motility due to flagellar defects

• Acrosomal abnormalities: Impaired fertilization capacity

Methodological approaches for translational research include:

• Genetic screening: Sequencing PFN4 in infertile men with matching phenotypes

• Expression analysis: Assessing PFN4 levels in testicular biopsies from infertile men

• Functional sperm assays: Evaluating acrosome reaction and fertilization capacity

Importantly, in vitro fertilization experiments with PFN4-deficient mouse sperm demonstrated successful fertilization of zona-free oocytes and development to the morula stage . This suggests potential treatment approaches for PFN4-related infertility through assistive reproductive technologies like intracytoplasmic sperm injection (ICSI).

How can researchers develop diagnostic approaches for PFN4-related male infertility?

Development of diagnostic tools for PFN4-related male infertility would require:

• Biomarker identification:

  • Sperm morphology parameters specifically associated with PFN4 deficiency

  • Molecular signatures detectable in semen samples

  • Immunological detection of PFN4 protein levels or modifications

• Genetic testing protocols:

  • Targeted sequencing of PFN4 and associated genes

  • Identification of clinically relevant variants

  • Functional validation of variants of unknown significance

• Phenotypic characterization:

  • Standardized assessment of manchette-related defects

  • Acrosome integrity evaluation

  • Correlation between PFN4 status and fertility outcomes

• Clinical validation:

  • Prospective studies in infertile populations

  • Genotype-phenotype correlation analysis

  • Treatment outcome prediction based on PFN4 status

How does PFN4 function compare across different species?

Comparative analysis of PFN4 across species provides valuable insights into its evolutionary conservation and functional specialization:

• Evolutionary conservation:
  PFN4 shows limited homology (approximately 30%) to other profilin family members, suggesting rapid evolutionary divergence and functional specialization .

• Expression patterns:
  The testis-specific expression appears conserved across mammals, indicating specialized reproductive functions.

• Functional conservation:
  Cross-species comparison of knockout phenotypes would reveal the degree of functional conservation and potential species-specific adaptations.

Methodological approaches for comparative studies include:

• Phylogenetic analysis of PFN4 across diverse species

• Functional complementation experiments with cross-species PFN4 variants

• Structural comparison of PFN4 proteins from different organisms

What can be learned from comparing phenotypes of different profilin-deficient models?

Comparative analysis of profilin knockout models provides context for understanding PFN4's specific functions:

These comparative phenotypes suggest evolutionary subfunctionalization within the profilin family, with PFN4 acquiring specialized roles in manchette formation and acrosome biogenesis essential for male fertility.

Researchers can leverage these comparisons through:

• Generation of compound knockout models

• Domain-swap experiments between different profilins

• Mechanistic studies of functional redundancy and specialization