ARPC5 Human

What is the basic structure and function of ARPC5 in humans?

ARPC5 is one of seven subunits of the Arp2/3 complex, which is essential for actin nucleation and the formation of branched actin networks. In humans, ARPC5 exists as two paralogous genes (ARPC5 and ARPC5L) that encode proteins with 67% sequence identity . The proteins encoded by these genes are structurally distinct, with the N-terminal half of ARPC5L being partially disordered compared to ARPC5 . This structural distinction likely contributes to differences in Arp2/3 complex activity when assembled with one or the other of these paralogs. ARPC5 has relatively low tissue and cell specificity in normal tissues, cell lines, and single-cell types .

The Arp2/3 complex containing ARPC5 participates in numerous cellular processes including:

• Cell migration and chemotaxis

• Immune cell function

• Developmental processes

• Cytoskeletal organization and remodeling

The presence of two ARPC5 paralogs allows for the formation of different "iso-complexes" of Arp2/3 with potentially distinct properties and biological roles .

What clinical phenotypes are associated with ARPC5 deficiency in humans?

ARPC5 deficiency results in a severe autosomal recessive primary immunodeficiency syndrome characterized by:

• Recurrent infections indicating impaired immune function

• Multiple congenital anomalies affecting neurological, cardiovascular, craniofacial, and hematopoietic development

• Gastrointestinal manifestations including diarrhea

• Thrombocytopenia

• Early postnatal death, often from sepsis

A case study identified a biallelic ARPC5 frameshift variant (c.189delT, p.(Ile67Serfs*8)) in a female child from consanguineous parents who presented with these symptoms . Her parents also had a previous child who died with similar clinical features, suggesting the same genetic cause. This phenotype overlaps with, but is more severe than, deficiency of ARPC1B, another subunit of the Arp2/3 complex .

How does ARPC5 differ from its paralog ARPC5L?

While ARPC5 and ARPC5L share 67% sequence identity, several key differences exist:

• Structural differences: The N-terminal half of ARPC5L is partially disordered compared to ARPC5, which may contribute to functional distinctions .

• Non-redundant function: Despite upregulation of ARPC5L in the absence of ARPC5, ARPC5L cannot compensate for ARPC5 deficiency in humans or mouse models, indicating non-redundant biological roles .

• Developmental importance: ARPC5 appears to be more critical for embryonic development than ARPC5L, as evidenced by the embryonic lethality in Arpc5−/− mice .

• Complex assembly impact: The presence of either ARPC5 or ARPC5L in the Arp2/3 complex likely results in "iso-complexes" with different properties and biological roles, similar to what has been observed with ARPC1 paralogs .

What are the molecular mechanisms by which ARPC5 deficiency affects cellular function?

ARPC5 deficiency disrupts cellular function through several interconnected mechanisms:

• Impaired actin cytoskeleton organization: CRISPR/Cas9-generated ARPC5-deficient HeLa cells exhibit dramatic reduction in cell spreading with loss of actin stress fibers and focal adhesions . This disorganization of the actin cytoskeleton underlies many of the downstream cellular defects.

• Reduced cell migration: ARPC5-deficient cells show significantly impaired migration capabilities. In experimental models, ARPC5−/− HeLa cells demonstrated reduced velocities (0.17±0.06 μm/min vs. 0.20±0.06 μm/min in wild-type) and displacements (10.39±0.94 μm vs. 15.27±3.26 μm in wild-type) . This migration defect likely contributes to developmental abnormalities and immune dysfunction.

• Disrupted chemokine-dependent migration: Studies using CRISPR/Cas9 approaches have demonstrated that loss of ARPC5 affects chemokine-dependent cell migration in vitro , which is particularly relevant for immune cell function.

• Impaired development: In mouse models, homozygous Arpc5−/− embryos fail to survive past embryonic day 9 due to severe developmental defects, including loss of the second pharyngeal arch which contributes to craniofacial and heart development . This suggests critical roles for ARPC5 in embryonic morphogenesis.

• Dysregulated immune signaling: ARPC5 appears important for postnatal immune signaling in a manner that cannot be compensated for by ARPC5L , though the precise molecular mechanisms require further elucidation.

What is the role of ARPC5 in cancer development and progression?

ARPC5 shows significant associations with cancer development and progression across multiple tumor types:

• Expression pattern: ARPC5 expression is upregulated in most cancer types compared to normal tissues . This overexpression pattern suggests a potential role in oncogenesis.

• Prognostic significance: High ARPC5 expression is significantly associated with worse prognosis in several cancers, particularly:

  • Kidney renal clear cell carcinoma (KIRC)

  • Kidney renal papillary cell carcinoma (KIRP)

  • Low-grade glioma (LGG)

  • Liver hepatocellular carcinoma (LIHC)

• Tumor microenvironment interactions: ARPC5 expression is positively correlated with tumor microenvironment scores and immune infiltrating cells in most cancers, suggesting a role in modulating the tumor immune landscape .

• Immune checkpoint association: ARPC5 expression correlates with immune checkpoint-related genes in most cancers, potentially influencing immunotherapy responses .

• Functional impact in hepatocellular carcinoma (HCC): Experimental studies have demonstrated that silencing ARPC5 dramatically decreases proliferation, migration, and invasion abilities of HCC cells . ARPC5 expression is significantly elevated in HCC tissues and cell lines compared to normal liver.

• Genetic instability markers: In some cancers (STAD and BRCA), ARPC5 positively correlates with tumor mutational burden (TMB), microsatellite instability (MSI), and neoantigens , suggesting potential roles in genomic instability or as a biomarker for immunotherapy response.

• Epigenetic interactions: ARPC5 expression correlates with various RNA modification genes (m1A-related, m5C-related, m6A-related) and DNA methyltransferases , indicating potential epigenetic regulatory mechanisms.

These findings suggest ARPC5 could serve as both a prognostic biomarker and potential therapeutic target in various cancers.

What evolutionary insights can be derived from comparing ARPC5 function across species?

Evolutionary analysis of ARPC5 reveals important insights about its conserved and divergent functions:

• Conservation of developmental function: The embryonic lethality of Arpc5−/− mice by embryonic day 9 contrasts with ARPC1B-deficient mice, which do not show impaired prenatal development or survival . This suggests that ARPC5's developmental role is more evolutionarily conserved and less redundant than that of other complex subunits.

• Paralog divergence: The functional non-redundancy between ARPC5 and ARPC5L in mammals represents an evolutionary adaptation that allows for specialized functions of the Arp2/3 complex. This paralog divergence mechanism appears to be a common theme among Arp2/3 complex subunits, as similar functional specialization has been observed between ARPC1A and ARPC1B .

• Species-specific subunit requirements: The variable phenotypic severity associated with deficiencies in different Arp2/3 complex subunits across species suggests evolutionary divergence in the regulatory networks controlling actin cytoskeleton organization.

• Conservation of immune function: The immunodeficiency phenotypes observed in both ARPC5- and ARPC1B-deficient humans highlight the evolutionarily conserved importance of proper actin cytoskeleton regulation in immune system function .

What are the optimal approaches for modeling ARPC5 deficiency in laboratory settings?

Several complementary approaches have proven effective for modeling ARPC5 deficiency:

• CRISPR/Cas9 gene editing: This has been successfully used to generate ARPC5-deficient cell lines (THP1 and HeLa) and mouse models . For cellular models:

  • Target guide RNAs to early exons for complete knockout

  • Verify knockout at both mRNA and protein levels

  • Assess potential compensatory upregulation of ARPC5L

  • Consider generating ARPC5/ARPC5L double knockout models for comparative studies

• Mouse models: Constitutive Arpc5−/− mice exhibit embryonic lethality by day 9, limiting their utility for postnatal studies . Alternative approaches include:

  • Conditional knockout models using tissue-specific Cre-lox systems

  • Heterozygous models to study gene dosage effects

  • Knockin models of human pathogenic variants

  • Inducible knockout systems for temporal control

• Patient-derived materials: When available, cells from patients with ARPC5 deficiency provide the most clinically relevant model. Consider:

  • Establishing immortalized cell lines

  • Generating induced pluripotent stem cells (iPSCs)

  • Deriving organoids for tissue-specific studies

• Paralog comparison studies: Include parallel experiments with ARPC5L-deficient models to dissect paralog-specific functions.

• Rescue experiments: Reintroduce wild-type or mutant ARPC5 into deficient models to establish causality and structure-function relationships.

How can researchers effectively assess actin cytoskeleton dynamics in ARPC5-deficient systems?

Comprehensive analysis of actin cytoskeleton dynamics in ARPC5-deficient systems requires multiple complementary approaches:

• Immunofluorescence imaging:

  • Phalloidin staining for F-actin visualization

  • Co-staining for focal adhesion markers (e.g., vinculin)

  • Quantification of stress fiber formation, cell spreading, and lamellipodia formation

• Live-cell imaging:

  • Fluorescently tagged actin (e.g., LifeAct) for real-time visualization

  • FRAP (Fluorescence Recovery After Photobleaching) to measure actin turnover rates

  • Particle tracking for quantitative assessment of actin flow

• Migration assays:

  • Wound healing/scratch assays

  • Transwell migration assays

  • Chemotaxis assays using chemokine gradients to assess directed migration

  • Time-lapse microscopy with cell tracking to quantify:

    • Velocity (μm/min)

    • Displacement (μm)

    • Directionality ratios

• Biochemical assays:

  • Actin polymerization assays

  • Arp2/3 complex assembly and activity assays

  • Fractionation to separate G-actin and F-actin pools

• Electron microscopy:

  • Scanning electron microscopy to visualize cell surface morphology

  • Transmission electron microscopy to examine actin filament organization

• Proteomic approaches:

  • Proximity labeling techniques to identify ARPC5 interaction partners

  • Phosphoproteomic analysis to assess signaling changes

  • Comparative proteomics between ARPC5- and ARPC5L-containing complexes

What experimental considerations are important when investigating the role of ARPC5 in immune cell function?

When investigating ARPC5's role in immune function, researchers should consider:

• Cell type selection:

  • Myeloid cells (neutrophils, macrophages, dendritic cells)

  • Lymphoid cells (T cells, B cells, NK cells)

  • Choose primary cells when possible, or appropriate cell lines (e.g., THP1) when necessary

• Functional assays:

  • Chemotaxis assays with relevant chemokines

  • Phagocytosis assays

  • Antigen presentation assays

  • Cytokine production measurements

  • Immunological synapse formation

• Signaling pathway analysis:

  • Receptor-mediated signaling cascades

  • Calcium flux measurements

  • Phosphorylation status of key immune signaling molecules

• In vivo considerations:

  • Immune challenge models in conditional knockout mice

  • Bone marrow chimeras to isolate hematopoietic contributions

  • Adoptive transfer experiments to assess cell-intrinsic effects

• Patient-derived materials:

  • When available, analyze immune cells from ARPC5-deficient patients

  • Compare with ARPC1B-deficient patient samples to identify subunit-specific effects

• Compensatory mechanisms:

  • Assess ARPC5L upregulation in ARPC5-deficient immune cells

  • Investigate potential alternative actin nucleation pathways

  • Consider redundancy with other actin regulatory proteins

• Temporal considerations:

  • Acute vs. chronic ARPC5 deficiency effects

  • Developmental vs. adult immune phenotypes

  • Age-dependent compensation mechanisms

What therapeutic strategies might target ARPC5 in cancer and immunological disorders?

Several potential therapeutic approaches targeting ARPC5 merit investigation:

• For cancer treatment:

  • Small molecule inhibitors specifically targeting ARPC5-containing Arp2/3 complexes

  • siRNA or antisense oligonucleotide approaches for ARPC5 silencing

  • Targeting ARPC5-dependent signaling pathways

  • Combination therapies with immune checkpoint inhibitors, given ARPC5's correlation with immune checkpoint genes

  • Biomarker development for patient stratification based on ARPC5 expression levels

• For ARPC5 deficiency disorders:

  • Gene therapy approaches to restore ARPC5 expression

  • Stem cell transplantation for immune reconstitution

  • Pharmacological enhancement of ARPC5L function to compensate for ARPC5 deficiency

  • Targeted therapies for specific downstream consequences (e.g., anti-inflammatory agents)

  • Prenatal genetic diagnosis for families with known ARPC5 mutations

• Common considerations:

  • Cell type-specific delivery systems to target relevant tissues

  • Temporal control of therapeutic intervention

  • Careful assessment of off-target effects

  • Monitoring for compensatory upregulation of ARPC5L or alternative pathways

How might single-cell technologies advance our understanding of ARPC5 function?

Single-cell technologies offer powerful approaches to dissect ARPC5 function:

• Single-cell RNA sequencing (scRNA-seq):

  • Characterize cell type-specific expression patterns of ARPC5 and ARPC5L

  • Identify cellular subpopulations differentially affected by ARPC5 deficiency

  • Map compensatory transcriptional networks in ARPC5-deficient cells

  • Track developmental trajectories in heterogeneous populations

• Single-cell ATAC-seq:

  • Identify regulatory elements controlling ARPC5 expression

  • Map chromatin accessibility changes in ARPC5-deficient cells

  • Discover potential transcription factors regulating ARPC5 expression

• Single-cell proteomics:

  • Quantify ARPC5 and ARPC5L protein levels at single-cell resolution

  • Identify protein interaction networks altered by ARPC5 deficiency

  • Measure phosphorylation states of signaling proteins downstream of ARPC5

• Spatial transcriptomics/proteomics:

  • Map ARPC5 expression patterns within tissues

  • Identify spatial relationships between ARPC5-expressing cells and their microenvironment

  • Visualize actin cytoskeletal changes in the context of tissue architecture

• Live-cell imaging at single-cell level:

  • Track dynamic changes in actin organization in individual cells

  • Correlate ARPC5 expression levels with phenotypic outcomes

  • Measure cell-to-cell variability in response to perturbations

What are the challenges in translating ARPC5 research findings to clinical applications?

Translating ARPC5 research to clinical applications faces several challenges:

• Genetic complexity:

  • Distinguishing pathogenic from benign variants in ARPC5

  • Assessing the impact of genetic background on ARPC5-related phenotypes

  • Developing appropriate genetic testing strategies for rare ARPC5-related disorders

• Functional redundancy:

  • Understanding the compensatory role of ARPC5L and its limitations

  • Identifying the unique and shared functions of ARPC5 paralogs

  • Developing therapeutic strategies that account for paralog-specific functions

• Developmental timing:

  • Determining critical developmental windows for ARPC5 function

  • Establishing appropriate timing for therapeutic interventions

  • Addressing prenatal developmental defects that may be irreversible

• Targeting specificity:

  • Developing drugs that specifically target ARPC5 without affecting ARPC5L

  • Achieving cell type-specific targeting to minimize side effects

  • Balancing inhibition in cancer contexts versus enhancement in deficiency disorders

• Diagnostic challenges:

  • Recognizing ARPC5 deficiency as a cause of syndromic immunodeficiency

  • Establishing standardized diagnostic criteria

  • Developing accessible testing methods for resource-limited settings

• Therapeutic delivery:

  • Developing effective delivery systems for gene therapy or protein replacement

  • Achieving sufficient therapeutic levels in relevant tissues

  • Navigating regulatory pathways for novel therapeutic modalities