BIN2 Human

What is BIN2 and what is its basic cellular function?

BIN2, also known as Breast cancer-associated protein 1 (BRAP1), is a membrane-sculpting N-BAR domain-containing protein highly expressed in leukocytic cells. Its primary functions involve membrane remodeling processes that influence cell motility, adhesion, and phagocytosis in leukocytes . BIN2 acts as a key regulator of actin-rich structures on the plasma membrane and is involved in podosome formation and dynamics . Unlike BRAP2 which has a similar name but unrelated sequence, BIN2 contains an N-terminal BAR domain and a C-terminus rich in glutamates, serines, and prolines that is heavily phosphorylated .

What is the structural composition of the BIN2 protein?

The structure of BIN2 consists of an N-terminal N-BAR domain and a C-terminus comprising 328 of the 565 amino acids in human BIN2. The N-BAR domain forms an elongated banana-shaped dimer, similar to other BAR domain proteins. Each monomer contains three long, bent helices with specific structural features :

• Helix 1: amino acids 38-88

• Helix 2: amino acids 95-159 (contains two kinks at Asp119 and Gln131)

• Helix 3: amino acids 169-238 (disrupted at Pro196)

• A 28 amino acid amphipathic helix (H0) at the N-terminus

The C-terminus is rich in glutamates (41), serines (58), and prolines (43) with no strong homology to other proteins and is the most divergent region across species .

How conserved is BIN2 across different species?

BIN2 shows variable conservation patterns between species, with the C-terminus being the most divergent region. Sequence analysis reveals:

• The C-terminus has only 43% sequence identity between human and rat

• Even lower conservation (15% sequence identity) exists between human and dog sequences

• The protein is found in vertebrates including humans, rat, dog, frog (Xenopus), and zebrafish (Danio)

• BIN2 homologs are not found in invertebrates such as worms or flies

The higher conservation of the N-BAR domain across species suggests evolutionary importance of the membrane-sculpting function, while the divergent C-terminus may reflect species-specific regulatory mechanisms.

What is the tissue distribution pattern of BIN2 in humans?

BIN2 demonstrates a specific tissue expression pattern in humans, being highly enriched in leukocytic cells. Expression data from the Human Protein Atlas reveals BIN2 presence across multiple tissues, with particularly notable expression in immune system-related tissues . The protein is endogenously expressed in:

• Mast cells (as shown in the RBL·2H3 cell line)

• B cells

• Macrophages

• Other leukocyte lineages

This restricted expression pattern suggests BIN2 has specialized functions in immune system cells rather than being a general cellular component.

What is the subcellular localization of BIN2 in leukocytes?

BIN2 demonstrates specific subcellular localization patterns in leukocytes, particularly associated with dynamic membrane structures. Live-cell imaging experiments have revealed that:

• BIN2 localizes to ring-like structures around the actin core of podosomes

• It associates with actin-rich structures on the plasma membrane

• It is found at the leading edge of migrating cells

• BIN2 is present in the macrophage phagocytic cup structure

• This localization is dependent on its N-BAR domain

The protein's targeting to these specific membrane regions supports its role in membrane sculpting and cytoskeletal regulation at sites of active membrane remodeling.

What experimental approaches can verify BIN2 subcellular localization?

To verify BIN2 subcellular localization, researchers can employ several complementary techniques:

• Fluorescence microscopy with:

  • Antibody-based immunofluorescence of endogenous BIN2

  • Expression of fluorescently-tagged BIN2 constructs (e.g., Bin2-EGFP)

  • Co-localization studies with known markers of cellular structures (e.g., actin)

• Domain mapping experiments:

  • Expression of truncated constructs (N-BAR domain only vs. C-terminus only)

  • Site-directed mutagenesis of key residues (especially in the amphipathic helix)

• Biochemical fractionation:

  • Separation of membrane and cytosolic fractions

  • Western blotting to detect BIN2 in different cellular compartments

Studies have shown that while full-length BIN2 and its N-BAR domain localize to podosomes, the C-terminus alone remains cytosolic, confirming the N-BAR domain's critical role in membrane targeting .

How does BIN2 interact with membranes and what structural features enable this interaction?

BIN2 interacts with membranes through its N-BAR domain, which has specialized structural features for membrane binding and sculpting:

• The curved "banana-shaped" BAR domain dimer that senses and/or induces membrane curvature

• A critical N-terminal amphipathic helix (H0) that:

  • Inserts into the membrane upon binding

  • Contains bulky hydrophobic residues (F13 and F21) essential for stable membrane interaction

  • Is more amphipathic than corresponding helices in related proteins like rEndoA1 and dAmph

  • When deleted or mutated (F13A/F21A), results in substantial loss of membrane binding capacity

• Positive charges on the concave surface that interact with negatively charged phospholipids

The membrane-binding mechanism involves both insertion of the amphipathic helix into the membrane and electrostatic interactions between the positively charged concave surface of the BAR domain and negatively charged lipids .

What protein-protein interactions does BIN2 engage in?

BIN2 engages in several specific protein-protein interactions, primarily through its C-terminal domain:

• SH3 domain-containing proteins:

  • α-PIX (also known as ARHGEF6) - direct binding confirmed by pull-down experiments

  • β-PIX (ARHGEF7)

  • Endophilin A2

• Git2 (G protein-coupled receptor kinase-interacting protein 2)

These interactions are likely mediated by:

• Two consensus PxxP sequences in the C-terminus (PTSPR at positions 445-449 and PEKPVR at positions 472-477)

• Additional polyproline regions that don't follow known consensus sequences

The PIX-Git complex is known to localize to the ring-like structure of podosomes, consistent with BIN2's localization . These protein interactions suggest BIN2 functions as an adaptor protein that connects membrane remodeling with signaling pathways that regulate cytoskeletal dynamics.

What is BIN2's lipid binding specificity?

BIN2 demonstrates preferential binding to phosphoinositide-enriched membranes, though without strong selectivity for specific phosphoinositide species. Lipid cosedimentation assays have shown:

• Higher affinity for phosphoinositide (PIP)-enriched membranes compared to membranes without PIPs

• No clear preference for particular PIP species was observed in the available studies

• This preference for PIP-enriched membranes is consistent with BIN2's localization to podosomes, which are known to be rich in phosphoinositides

The interaction with phosphoinositide-rich membranes is likely important for BIN2's specific targeting to particular membrane domains in the cell, such as podosomes where PI3K signaling is necessary for formation .

What role does BIN2 play in podosome formation and dynamics?

BIN2 is a critical regulator of podosome biology in leukocytes. Experimental manipulation of BIN2 expression reveals its specific impacts:

• Upon BIN2 knockdown using siRNA:

  • Reduced podosome density

  • Decreased podosome dynamics

  • Podosomes still form but are altered in number and behavior

• With BIN2 overexpression:

  • Increased podosome density

  • Enhanced podosome dynamics

• Mechanistic insights:

  • BIN2 localizes to the ring-like structure surrounding the actin core of podosomes

  • This localization depends on its N-BAR domain

  • BIN2 likely serves as a scaffold that connects membrane remodeling to cytoskeletal dynamics

  • Its interactions with α-PIX and other proteins at podosomes suggest involvement in signaling pathways that regulate actin dynamics

These findings establish BIN2 as an important component of the podosome regulatory machinery, influencing both the formation and dynamic behavior of these structures in leukocytes.

How does BIN2 influence cell migration in leukocytes?

BIN2 plays a significant role in regulating leukocyte migration, as demonstrated by loss-of-function studies:

• siRNA-mediated knockdown of endogenous BIN2 results in:

  • Decreased monocyte migration capacity

  • Altered cell morphology during migration

  • Potential disruption of the coordination between membrane remodeling and cytoskeletal dynamics required for efficient migration

• Mechanistic basis:

  • The localization of BIN2 to the leading edge of migrating cells suggests its involvement in membrane protrusion

  • Its interaction with α-PIX, which regulates Rac1 and Cdc42 GTPases, links BIN2 to pathways controlling actin polymerization and lamellipodial/filopodial formation

  • The membrane sculpting activity of BIN2 may contribute to the membrane deformations necessary for migration

These findings indicate that BIN2 is an important component of the molecular machinery driving leukocyte migration, potentially coordinating membrane remodeling with cytoskeletal dynamics.

What is the relationship between BIN2 expression and phagocytic function?

BIN2 exhibits a complex relationship with phagocytic function in leukocytes, with expression level having opposite effects on phagocytosis:

• BIN2 knockdown effects:

  • Increased phagocytosis in macrophages

  • Suggests BIN2 normally functions as a negative regulator of phagocytosis

• BIN2 overexpression effects:

  • Decreased phagocytic capacity

  • Reinforces its role as a restraint on phagocytic activity

• Mechanistic insights:

  • BIN2 localizes to the macrophage phagocytic cup

  • Its membrane sculpting activity may influence phagocytic cup formation and closure

  • The inverse relationship between BIN2 expression and phagocytosis suggests it may regulate the temporal or spatial coordination of membrane remodeling during phagocytosis

These findings establish BIN2 as a negative regulator of phagocytosis while simultaneously being required for efficient cell migration, highlighting its multifaceted role in leukocyte function.

What methods are optimal for studying BIN2 membrane interactions in vitro?

Several complementary methods can be employed to study BIN2 membrane interactions in vitro:

• Lipid cosedimentation assays:

  • Mix purified BIN2 protein with liposomes of defined composition

  • Centrifuge to separate membrane-bound and free protein

  • Analyze fractions by SDS-PAGE/Western blotting

  • Useful for quantifying binding affinity and lipid preferences

• Electron microscopy of membrane tubulation:

  • Incubate liposomes with purified BIN2

  • Visualize membrane tubulation activity using negative staining EM

  • Assess the diameter and morphology of induced tubules

• Structure-function analyses:

  • Generate point mutations in key residues (e.g., F13A/F21A in the amphipathic helix)

  • Assess impact on membrane binding and tubulation

  • Correlate structural features with functional outcomes

• Fluorescence microscopy with giant unilamellar vesicles (GUVs):

  • Visualize BIN2-membrane interactions in real-time

  • Use fluorescently labeled protein and/or lipids

  • Observe membrane deformation directly

These approaches have successfully revealed BIN2's membrane binding properties, including its requirement for an intact amphipathic helix and preference for phosphoinositide-enriched membranes.

What cellular models are appropriate for studying BIN2 function?

Based on BIN2's expression pattern and functions, the following cellular models are appropriate for studying its biology:

• Myeloid cell lines:

  • THP-1 cells (human monocytic cell line)

  • RAW264.7 cells (mouse macrophage line)

  • RBL·2H3 cells (rat basophilic leukemia cells, a mast cell model)

• B cell models:

  • Various B cell lines that endogenously express BIN2

• Primary cells:

  • Human peripheral blood monocytes

  • Bone marrow-derived macrophages

  • Dendritic cells

• Functional assays in these models:

  • Migration assays (transwell, wound healing)

  • Phagocytosis assays (fluorescent bead uptake, bacterial phagocytosis)

  • Podosome formation and dynamics (live-cell imaging)

  • Immunofluorescence for protein localization

Selection of the appropriate model system should be based on the specific aspect of BIN2 function being investigated and the availability of tools to manipulate BIN2 expression in that system.

What genetic manipulation approaches are effective for BIN2 functional studies?

Multiple genetic manipulation strategies have proven effective for BIN2 functional studies:

• RNA interference:

  • siRNA approaches successfully reduce endogenous BIN2 expression

  • Allow assessment of loss-of-function phenotypes in various cellular processes

• Overexpression systems:

  • Full-length BIN2-EGFP fusions enable visualization of protein localization

  • Domain-specific constructs (N-BAR domain, C-terminus) help dissect domain functions

  • siRNA-insensitive constructs permit rescue experiments in knockdown cells

• Structure-based mutagenesis:

  • Point mutations in key residues (e.g., F13A/F21A in the amphipathic helix)

  • Mutations in dimerization interfaces

  • Alterations to protein-protein interaction motifs (e.g., PxxP motifs)

• CRISPR/Cas9 genome editing:

  • Generation of knockout cell lines

  • Introduction of endogenous tags for visualization of native protein

These approaches have been successfully employed to demonstrate BIN2's roles in membrane binding, podosome dynamics, cell migration, and phagocytosis.

How might BIN2's membrane sculpting activity be regulated by post-translational modifications?

BIN2's activity is likely regulated by post-translational modifications, particularly phosphorylation:

• Potential phosphoregulation mechanisms:

  • The C-terminus of BIN2 is heavily phosphorylated according to phosphosite.org data

  • Contains 58 serine residues that may serve as phosphorylation sites

  • Phosphorylation could alter:

    • Protein-protein interactions

    • Conformational dynamics

    • Membrane binding affinity

    • Subcellular localization

• Research approaches to investigate phosphoregulation:

  • Mass spectrometry to identify phosphorylation sites

  • Phosphomimetic and phosphodeficient mutations

  • Kinase inhibitor studies to identify relevant signaling pathways

  • Correlation of phosphorylation status with functional outcomes

• Potential kinases involved:

  • Given BIN2's role in immune cells, candidates include:

    • PKC isoforms activated during immune cell stimulation

    • Src family kinases involved in immunoreceptor signaling

    • MAP kinases that respond to inflammatory signals

Understanding the phosphoregulation of BIN2 would provide insights into how its membrane sculpting activities are dynamically controlled during immune cell functions.

What is the interplay between BIN2 and the actin cytoskeleton in leukocyte function?

The relationship between BIN2 and the actin cytoskeleton represents an important area for advanced investigation:

• Current evidence of interplay:

  • BIN2 localizes to actin-rich structures including podosomes and the leading edge

  • BIN2 influences podosome dynamics, which require coordinated actin remodeling

  • BIN2 interacts with proteins (α-PIX, Git2) that regulate actin dynamics through Rho GTPases

• Potential mechanisms of coordination:

  • BIN2 may temporally or spatially regulate membrane deformation to facilitate actin polymerization

  • The membrane sculpting activity may create membrane curvature that recruits or activates actin regulators

  • BIN2's interactions with SH3 domain-containing proteins might assemble signaling complexes that regulate actin dynamics

• Advanced research questions:

  • Does BIN2 directly or indirectly influence actin polymerization rates?

  • How does BIN2 coordinate with actin during specific processes like podosome formation or phagocytic cup closure?

  • What is the temporal relationship between BIN2 recruitment and actin remodeling during leukocyte activation?

Resolving these questions would enhance our understanding of how membrane remodeling and cytoskeletal dynamics are coordinated during complex leukocyte functions.

What are the potential implications of BIN2 in immune cell-related pathologies?

Given BIN2's role in fundamental leukocyte functions, it may have implications in various immune cell-related pathologies:

• Potential involvement in inflammatory diseases:

  • Dysregulated migration of leukocytes contributes to inflammatory pathologies

  • Altered BIN2 expression or function might impact:

    • Leukocyte recruitment to inflammatory sites

    • Transendothelial migration

    • Interstitial motility within inflamed tissues

• Possible roles in infectious disease:

  • BIN2's negative regulation of phagocytosis suggests it might influence:

    • Clearance of pathogens

    • Resolution of infection

    • Balance between protective and pathological inflammation

• Implications in cancer:

  • BIN2 was originally identified as Breast cancer-associated protein 1 (BRAP1)

  • Its effects on cell migration might influence:

    • Tumor-associated macrophage function

    • Immune cell infiltration into tumors

    • Potentially the migration of cancer cells that aberrantly express BIN2

• Research approaches needed:

  • Expression analysis in patient samples from relevant diseases

  • Correlation of expression/mutation status with disease outcomes

  • Development of animal models with tissue-specific BIN2 manipulation

Understanding BIN2's role in these pathologies could potentially identify it as a therapeutic target for modulating specific leukocyte functions in disease contexts.

How do in vitro and cellular approaches to studying BIN2 complement each other?

A comprehensive understanding of BIN2 function requires integration of both in vitro biochemical and cellular approaches:

The integration of these complementary approaches has been crucial for establishing BIN2 as a bona fide membrane-sculpting protein with important roles in leukocyte function .

What are the most significant methodological challenges in studying BIN2 function?

Several methodological challenges complicate the comprehensive study of BIN2 function:

• Structural challenges:

  • The intrinsically disordered C-terminal region (328 of 565 amino acids) resists crystallization

  • Full-length protein structure determination remains elusive

  • Understanding how the structured N-BAR domain and disordered C-terminus coordinate is difficult

• Dynamic membrane interactions:

  • Capturing transient membrane interactions during cellular processes is technically challenging

  • Distinguishing BIN2's direct effects on membranes from indirect effects via binding partners

  • Quantifying membrane curvature changes in living cells with sufficient resolution

• Redundancy and compensation:

  • Potential functional overlap with other BAR domain proteins

  • Compensatory mechanisms that mask phenotypes in loss-of-function studies

  • Difficulty in distinguishing primary from secondary effects

• Tissue-specific contexts:

  • Adapting experimental systems to different leukocyte types

  • Capturing the diversity of membrane environments across immune cell states

  • Translating findings between species given the divergence in the C-terminus

Addressing these challenges will require continued development of advanced imaging techniques, improved structural biology approaches for flexible proteins, and sophisticated genetic models that can overcome functional redundancy.

What emerging technologies might advance our understanding of BIN2 function?

Several cutting-edge technologies hold promise for deepening our understanding of BIN2 biology:

• Advanced imaging approaches:

  • Super-resolution microscopy (STORM, PALM) to visualize BIN2-membrane interactions beyond the diffraction limit

  • Lattice light-sheet microscopy for long-term 3D imaging of BIN2 dynamics with minimal phototoxicity

  • Correlative light and electron microscopy (CLEM) to connect BIN2 localization with ultrastructural membrane features

• Structural biology innovations:

  • Cryo-electron tomography of cellular structures containing BIN2

  • Integrative structural biology combining crystallography, NMR, and computational approaches for full-length protein

  • Single-molecule studies of BIN2-membrane interactions

• Genome editing and screening:

  • CRISPR-based screens to identify genetic interactors of BIN2

  • Endogenous tagging of BIN2 with split fluorescent proteins to visualize protein-protein interactions

  • Domain-specific knockouts to dissect function in vivo

• Systems biology approaches:

  • Proteomics of BIN2 interactome under different cellular states

  • Phosphoproteomics to comprehensively map BIN2 post-translational modifications

  • Integration of multi-omics data to place BIN2 in broader signaling networks

These technologies could overcome current limitations in understanding BIN2's dynamics, interactions, and regulatory mechanisms across different cellular contexts.

What are the potential therapeutic implications of manipulating BIN2 function?

Targeting BIN2 could have therapeutic potential in several disease contexts:

• Inflammatory diseases:

  • Modulating BIN2 to decrease leukocyte migration might reduce inflammatory cell infiltration

  • Targeting BIN2-protein interactions could fine-tune rather than abolish immune cell functions

  • Relevant contexts include rheumatoid arthritis, inflammatory bowel disease, and psoriasis

• Infection and immunity:

  • Enhancing phagocytic function by inhibiting BIN2 could improve bacterial clearance

  • Balancing migration and phagocytosis by selective BIN2 modulation might optimize immune responses

  • Particularly relevant for chronic or recurrent infections

• Cancer immunotherapy:

  • Manipulating BIN2 in tumor-associated macrophages could alter their phenotype or function

  • Enhancing immune cell infiltration into tumors by targeting BIN2

  • Potential relevance given BIN2's original identification as Breast cancer-associated protein 1 (BRAP1)

• Therapeutic approaches:

  • Small molecule inhibitors of BIN2-membrane interactions

  • Peptide-based disruptors of specific protein-protein interactions

  • Cell-type specific delivery strategies to target BIN2 in specific leukocyte populations

Development of such therapeutics would require deeper understanding of BIN2's tissue-specific functions and careful assessment of potential side effects on normal immune function.

How might BIN2 function integrate with broader immune signaling networks?

Understanding how BIN2 integrates with immune signaling networks represents an important frontier:

• Potential signaling connections:

  • Through its interaction with α-PIX, BIN2 may connect to Rac1/Cdc42 signaling pathways

  • The Git2 interaction suggests links to focal adhesion signaling via paxillin

  • Endophilin A2 binding may connect BIN2 to endocytic pathways

  • Phosphoinositide binding likely integrates BIN2 with PI3K signaling networks

• Immune receptor pathways:

  • BIN2 may function downstream of various immune receptors that trigger cytoskeletal remodeling

  • Candidates include:

    • Fc receptors involved in phagocytosis

    • Chemokine receptors that drive migration

    • Pattern recognition receptors that activate innate immune responses

• Research approaches needed:

  • Phosphoproteomic analysis after immune receptor stimulation

  • BIN2 interactome studies under different activation conditions

  • Live imaging of BIN2 dynamics during receptor triggering

  • Computational modeling of signaling networks incorporating BIN2 functions

Integrating BIN2 into broader immune signaling maps would advance our understanding of how membrane remodeling is coordinated with other cellular processes during immune responses.