FCHO1 Antibody, FITC conjugated

What is FCHO1 and what is its primary function in cellular processes?

FCHO1 (F-BAR domain only protein 1) is a key molecule involved in the early stages of clathrin-mediated endocytosis (CME). It functions by participating in membrane binding and bending, while also recruiting proteins essential for the formation of functional clathrin-coated pits . FCHO1 contains an F-BAR domain that interacts with the plasma membrane and a μ-homology domain (μHD) that facilitates protein-protein interactions, particularly with EPS15 and EPS15R . The protein plays a critical role in nucleating clathrin-coated vesicles at the plasma membrane and has been implicated in regulating Bmp signaling by mediating endocytosis of Bmp receptors . Additionally, recent research has identified FCHO1's involvement in T-cell proliferation and activation through its effect on T-cell receptor (TCR) clustering and internalization .

What applications is the FITC-conjugated FCHO1 polyclonal antibody suitable for?

The FITC-conjugated FCHO1 polyclonal antibody is suitable for multiple research applications, providing flexibility for various experimental designs. Primary applications include:

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of FCHO1 protein in solution-based samples .

• Immunocytochemistry/Immunofluorescence (ICC/IF): For visualization of FCHO1 localization within cells, where the FITC conjugation provides direct fluorescent detection without requiring secondary antibodies .

• Immunohistochemistry on paraffin-embedded tissues (IHC-P): For examination of FCHO1 expression patterns in tissue sections .

The antibody has been validated specifically for human samples, making it appropriate for studies involving human cell lines, primary cells, or tissue specimens .

What is the immunogen used to generate the FCHO1 polyclonal antibody?

The FITC-conjugated FCHO1 polyclonal antibody available from Nordic Biosite is generated using recombinant human F-BAR domain only protein 1, specifically amino acids 45-180, as the immunogen . This region contains part of the F-BAR domain, which is crucial for the protein's membrane interaction capabilities. The Abcam antibody (ab272640), while not specified as FITC-conjugated in the search results, uses a different immunogen corresponding to a recombinant fragment within human FCHO1 amino acids 250-400 . Understanding the specific immunogen used is essential for predicting epitope recognition and potential cross-reactivity with FCHO1 domains or related proteins.

How should FCHO1 antibodies be stored to maintain optimal activity?

For optimal preservation of antibody activity, the FITC-conjugated FCHO1 polyclonal antibody should be shipped at 4°C, and upon receipt, stored at either -20°C (for short-term storage) or -80°C (for long-term storage) . Repeated freeze-thaw cycles should be avoided as they can compromise antibody integrity and function. The antibody is typically provided in a stabilizing buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4 . This formulation helps maintain antibody stability during storage. When working with the antibody, it's advisable to aliquot it into smaller volumes to minimize freeze-thaw cycles and extend its functional lifespan.

How does FCHO1 dysfunction impact T-cell development and immune function?

Research has uncovered a critical role for FCHO1 in human T-cell development and immune function. Studies have identified ten unrelated patients with variable T and B cell lymphopenia who are homozygous for six distinct mutations in the FCHO1 gene . These mutations either lead to protein mislocalization or prevent FCHO1's interaction with binding partners like EPS15 and EPS15R .

The immunological impact of FCHO1 deficiency includes:

• Variable degrees of T- and B-cell lymphopenia

• Hypogammaglobulinemia

• Compromised T-cell responses to T-cell receptor (TCR) triggering

• Impaired TCR internalization in FCHO1-deficient T cells

Clinical manifestations in FCHO1-deficient patients include recurrent bacterial, viral, and fungal infections, with some developing diffuse large B-cell lymphoma (DLBCL) . The table below summarizes the immunological and clinical findings in patients with FCHO1 mutations:

This evidence establishes FCHO1 as essential for normal T-cell differentiation and function, linking CME to the human immune system's development and operation .

What are the molecular mechanisms by which FCHO1 mutations disrupt clathrin-mediated endocytosis?

FCHO1 mutations disrupt clathrin-mediated endocytosis through several molecular mechanisms, which have been elucidated through functional studies of patient-derived mutations. These mechanisms include:

These molecular defects ultimately result in perturbed clathrin-mediated endocytosis in multiple tissues, with particular functional consequences for T-cell receptor internalization and signaling .

How can FCHO1 antibodies be used to study the spatiotemporal dynamics of clathrin-coated pit formation?

FITC-conjugated FCHO1 antibodies can be powerful tools for investigating the spatiotemporal dynamics of clathrin-coated pit formation through several advanced microscopy approaches:

• Live cell imaging: Using membrane-permeable FITC-conjugated FCHO1 antibodies or antibody fragments in live cells allows for real-time visualization of FCHO1 recruitment to nascent endocytic sites. This approach can reveal the temporal sequence of FCHO1 engagement relative to other endocytic proteins .

• Co-localization studies: FITC-conjugated FCHO1 antibodies can be used in conjunction with antibodies against other endocytic proteins (like clathrin, AP-2, or EPS15) labeled with spectrally distinct fluorophores to analyze their spatial relationship during CCP formation .

• Super-resolution microscopy: Techniques like STORM, PALM, or STED microscopy combined with FITC-conjugated FCHO1 antibodies can provide nanoscale resolution of FCHO1 localization within the endocytic machinery, revealing structural details beyond the diffraction limit.

• FRAP (Fluorescence Recovery After Photobleaching): This technique can measure the dynamics of FCHO1 association with and dissociation from CCPs, providing insights into the protein's residency time and mobility during endocytosis.

Research has shown that FCHO1 is an early-arriving protein at sites of CCP formation, along with Eps15/R . The FCHO1 protein contains a membrane-binding N-terminal EFC/F-BAR domain and an adjoining intrinsically disordered linker that functions as an allosteric activator, driving the reconfiguration of AP-2 from the closed to the membrane-bound open state . Antibody-based imaging approaches can help elucidate these dynamics in various cell types and under different experimental conditions.

What is the relationship between FCHO1 and T-cell receptor (TCR) internalization?

FCHO1 plays a crucial role in T-cell receptor (TCR) internalization, which has significant implications for T-cell activation and function. The relationship between FCHO1 and TCR internalization can be characterized as follows:

• TCR clustering regulation: FCHO1 affects TCR clustering upon receptor triggering, which is a critical early step in T-cell activation. The absence of functional FCHO1 disrupts this process .

• Internalization mechanism: FCHO1-deficient T cells show severely perturbed internalization of the TCR receptor after stimulation. This defect can be rescued by the expression of wild-type FCHO1, confirming FCHO1's direct role in this process .

• T-cell activation consequences: Patient T cells lacking functional FCHO1 are unresponsive to TCR triggering, indicating that FCHO1-mediated TCR internalization is required for proper T-cell activation .

• Signaling pathway integration: FCHO1's role in TCR internalization connects clathrin-mediated endocytosis to T-cell signaling pathways, explaining how defects in a general cellular process like CME can lead to T-cell-specific immune deficiencies .

This relationship between FCHO1 and TCR internalization highlights the importance of endocytic processes in immune cell function and provides a mechanistic explanation for the T-cell deficiencies observed in patients with FCHO1 mutations .

What optimization steps are necessary when using FITC-conjugated FCHO1 antibodies for immunofluorescence studies?

When using FITC-conjugated FCHO1 antibodies for immunofluorescence studies, several optimization steps are essential to obtain specific, high-quality results:

• Fixation method selection: Different fixation methods (paraformaldehyde, methanol, acetone) can affect epitope accessibility. For FCHO1 detection, compare fixation methods to determine which best preserves the target epitope while maintaining cellular morphology.

• Permeabilization optimization: Adjust permeabilization conditions (agent type, concentration, and exposure time) to ensure antibody access to intracellular FCHO1 while preserving subcellular structures. Typical agents include Triton X-100, saponin, or digitonin at concentrations of 0.1-0.5%.

• Blocking protocol development: Implement thorough blocking (typically 5-10% serum, BSA, or commercial blocking buffers) to minimize background fluorescence, which is particularly important for FITC due to its somewhat higher autofluorescence compared to other fluorophores.

• Antibody concentration titration: Test a range of antibody dilutions to determine the optimal concentration that provides specific FCHO1 labeling with minimal background. For FITC-conjugated antibodies, higher dilutions may be necessary to avoid excessive background.

• Incubation conditions adjustment: Optimize antibody incubation time (typically 1-24 hours) and temperature (4°C, room temperature, or 37°C) to enhance specific binding while minimizing non-specific interactions.

• Counterstain compatibility: When selecting nuclear and other organelle counterstains, choose fluorophores with emission spectra distinct from FITC (excitation ~495 nm, emission ~519 nm) to avoid spectral overlap.

• Photobleaching prevention: Implement anti-fade reagents in mounting media and minimize exposure to light during processing and imaging to preserve FITC signal, as it is somewhat susceptible to photobleaching.

• Positive and negative controls: Include positive controls (cells/tissues known to express FCHO1) and negative controls (antibody isotype controls, FCHO1 knockdown cells) to validate staining specificity.

For co-localization studies with clathrin, adaptin, or EPS15, as described in the research literature , sequential staining protocols may be necessary to avoid antibody cross-reactivity.

How can researchers validate the specificity of FCHO1 antibodies in their experimental systems?

Validating the specificity of FCHO1 antibodies is crucial for ensuring reliable experimental results. Researchers should implement the following comprehensive validation approaches:

• Western blot analysis: Perform western blotting to confirm that the antibody detects a protein of the expected molecular weight (~95 kDa for full-length FCHO1). Compare protein lysates from cells with known FCHO1 expression levels, including wild-type and FCHO1 knockout or knockdown cells.

• Immunoprecipitation followed by mass spectrometry: Use the FCHO1 antibody for immunoprecipitation, then analyze the precipitated proteins by mass spectrometry to confirm FCHO1 identity and detect potential cross-reactive proteins.

• Genetic validation approaches:

  • Compare staining patterns in wild-type versus FCHO1 knockout cells generated using CRISPR-Cas9

  • Test antibody reactivity in cells transfected with FCHO1 siRNA versus control siRNA

  • Analyze cells overexpressing tagged FCHO1 constructs to confirm co-localization with antibody staining

• Cross-validation with multiple antibodies: Compare staining patterns obtained with the FITC-conjugated FCHO1 polyclonal antibody to those obtained with other validated FCHO1 antibodies targeting different epitopes.

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide (amino acids 45-180 for the Nordic Biosite antibody ) before staining to demonstrate that specific binding can be blocked.

• FCHO1 mutant analysis: Test antibody reactivity with cells expressing different FCHO1 mutants (such as p.R679P, p.A34P, and p.Stop687) to verify epitope specificity and detect potential alterations in staining patterns, as described in published research .

• Co-localization with known interaction partners: Confirm that antibody staining shows expected co-localization patterns with known FCHO1 interaction partners such as EPS15, EPS15R, and adaptin in wild-type cells but altered patterns in cells expressing FCHO1 mutants .

Implementing multiple validation approaches provides robust evidence for antibody specificity and increases confidence in experimental findings.

What protocols are recommended for studying FCHO1-mediated clathrin-coated pit formation?

For studying FCHO1-mediated clathrin-coated pit formation, the following protocols are recommended based on published research methodologies:

• Live-cell imaging of CCP dynamics:

  • Transfect cells with fluorescently tagged FCHO1 (wild-type or mutant) and clathrin light chain (CLC)

  • Use RFP-tagged CLC and GFP-tagged FCHO1 for dual-color imaging

  • Employ total internal reflection fluorescence (TIRF) microscopy to visualize events at the plasma membrane

  • Acquire images at 1-5 second intervals for 5-10 minutes

  • Analyze trajectories of CCP formation, maturation, and internalization using particle tracking software

• Quantitative co-localization analysis:

  • Fix cells expressing FCHO1 constructs using 4% paraformaldehyde

  • Immunostain for endogenous clathrin, AP-2, EPS15, or other endocytic proteins

  • Acquire high-resolution confocal z-stacks

  • Measure co-localization using Pearson's correlation coefficient or Manders' overlap coefficient

  • Compare wild-type FCHO1 to mutant variants (p.A34P, p.R679P, p.Stop687) to assess functional impact

• Biochemical analysis of protein interactions:

  • Perform co-immunoprecipitation experiments with wild-type or mutant FCHO1

  • Use tagged constructs (e.g., GFP-FCHO1) or antibodies against endogenous proteins

  • Probe for interaction partners (EPS15, EPS15R, AP-2 components)

  • Analyze results by western blotting or mass spectrometry

• Functional endocytosis assays:

  • For T-cell studies: Measure TCR internalization using fluorescently labeled anti-CD3 antibodies

  • For general endocytosis: Use established cargo proteins such as transferrin or epidermal growth factor

  • Compare endocytosis rates in cells expressing wild-type versus mutant FCHO1

  • Flow cytometry or microscopy-based approaches can quantify internalization kinetics

• Membrane recruitment assay:

  • Express truncated FCHO1 constructs (e.g., GFP-FCHO1 (1-609)) to assess the role of specific domains

  • Measure the restoration of AP-2 puncta formation in FCHO1-deficient cells

  • Quantify size, number, and intensity of AP-2-positive structures by image analysis

These protocols have been successfully employed to characterize FCHO1's role in CCP formation and to understand the functional consequences of disease-associated mutations .

How can researchers differentiate between the effects of FCHO1 and other F-BAR domain-containing proteins in endocytosis?

Differentiating between the specific effects of FCHO1 and other F-BAR domain-containing proteins in endocytosis requires strategic experimental approaches that isolate FCHO1-specific functions. Researchers can implement the following methodologies:

• Domain-specific perturbation:

  • Generate chimeric constructs swapping the F-BAR domains between FCHO1 and other F-BAR proteins

  • Create point mutations in conserved versus divergent residues within the F-BAR domain

  • Express isolated domains (F-BAR only, linker regions, μHD domains) to identify domain-specific functions

  • Analyze functional rescue capabilities of these constructs in FCHO1-deficient backgrounds

• Temporal dynamics analysis:

  • Perform high-resolution time-lapse imaging to determine the precise temporal sequence of recruitment for different F-BAR proteins to endocytic sites

  • Use optogenetic tools to acutely inactivate or recruit specific F-BAR proteins and assess immediate consequences

  • FRAP analysis can reveal differences in residence time and exchange rates between FCHO1 and other F-BAR proteins

• Interactome-specific approaches:

  • Perform comparative interactome analysis of FCHO1 versus other F-BAR proteins using BioID, proximity labeling, or co-immunoprecipitation coupled with mass spectrometry

  • Focus on unique interaction partners of FCHO1, such as its specific binding to EPS15/R

  • Use the μHD domain of FCHO1, which is not present in many other F-BAR proteins, as a distinguishing feature for interaction studies

• Cargo-specific endocytosis assays:

  • Compare the effects of FCHO1 depletion versus depletion of other F-BAR proteins on the internalization of different cargo molecules

  • For T-cell studies, specifically analyze TCR internalization, which has been linked to FCHO1 function

  • Quantify endocytosis of different receptor types to identify cargo-specific requirements for FCHO1

• Compensatory mechanism analysis:

  • In FCHO1-deficient cells, assess whether overexpression of other F-BAR proteins (e.g., FCHO2, Toca-1, FBP17) can rescue endocytic defects

  • Perform sequential and combined knockdowns of multiple F-BAR proteins to detect synergistic or redundant functions

  • Generate cell lines with endogenously tagged F-BAR proteins to monitor compensatory upregulation after FCHO1 depletion

• Patient-derived cell studies:

  • Utilize cells from patients with FCHO1 mutations to study endocytic defects in a physiologically relevant context

  • Compare these phenotypes to artificially generated deficiencies in other F-BAR proteins

  • Perform rescue experiments with various F-BAR proteins to determine specificity

Research has shown that FCHO1 has both overlapping and distinct functions from other F-BAR proteins. For example, the FCHO1 linker region specifically acts as an allosteric activator of AP-2, enabling its transition from the closed to open conformation, which is a distinctive function not shared by all F-BAR proteins .

What factors might cause high background when using FITC-conjugated FCHO1 antibodies?

When using FITC-conjugated FCHO1 antibodies, several factors can contribute to high background signal, complicating data interpretation. Understanding and addressing these factors is essential for obtaining clear, specific staining:

• Suboptimal fixation and permeabilization:

  • Overfixation can cause autofluorescence and increase non-specific binding

  • Excessive permeabilization may allow antibody access to normally inaccessible epitopes

  • Solution: Test different fixation times and permeabilization agent concentrations; consider comparing paraformaldehyde, methanol, and acetone fixation methods

• Insufficient blocking:

  • Inadequate blocking allows non-specific antibody binding to Fc receptors or charged cellular components

  • Solution: Increase blocking time (1-2 hours) and concentration (5-10% normal serum or BSA); consider adding 0.1-0.3% Triton X-100 or 0.05% Tween-20 to blocking buffer

• FITC-specific issues:

  • FITC has higher autofluorescence than some other fluorophores

  • FITC is sensitive to photobleaching and pH changes

  • Solution: Use longer excitation wavelengths during acquisition, work in slightly alkaline conditions (pH 8.0-8.5), and include anti-fade agents in mounting media

• Antibody concentration:

  • Too high antibody concentration increases non-specific binding

  • Solution: Perform a titration experiment to determine optimal antibody concentration; typically start with 1:100 dilution and test serial dilutions up to 1:1000

• Sample autofluorescence:

  • Cellular components like NADH, flavins, and lipofuscin naturally fluoresce in the FITC channel

  • Solution: Use spectral unmixing during image acquisition, treat samples with Sudan Black B (0.1-0.3%) or CuSO₄ (1-10 mM) to quench autofluorescence

• Cross-reactivity:

  • Polyclonal antibodies may recognize epitopes on proteins other than FCHO1

  • Solution: Pre-absorb antibody with tissue/cell lysates, use peptide competition assays, or include FCHO1-deficient samples as negative controls

• Inappropriate washing:

  • Insufficient washing leaves unbound antibody in the sample

  • Solution: Increase number and duration of washes (3-5 washes of 5-10 minutes each) with PBS containing 0.05-0.1% Tween-20

• Mounting media issues:

  • Incompatible mounting media can increase background or reduce signal-to-noise ratio

  • Solution: Use aqueous mounting media with anti-fade agents specifically formulated for FITC; avoid media with high autofluorescence

When studying FCHO1's colocalization with clathrin or other endocytic proteins, it's particularly important to optimize these parameters, as the punctate staining pattern characteristic of clathrin-coated pits can be obscured by high background .

How can researchers optimize detection of FCHO1 in T cells for immunological studies?

Optimizing FCHO1 detection in T cells for immunological studies requires specialized approaches due to T cells' small size, high nucleus-to-cytoplasm ratio, and the potentially low abundance of FCHO1. The following optimization strategies are recommended:

• T cell preparation and fixation:

  • For primary T cells: Isolate using negative selection to avoid activating antibodies bound to cell surface

  • Optimize adhesion to slides/coverslips using poly-L-lysine or Cell-Tak coating

  • Fix cells with 2-4% paraformaldehyde for 10-15 minutes at room temperature

  • Gentle permeabilization with 0.1% saponin is often preferred for T cells to preserve membrane structures

• T cell activation state considerations:

  • FCHO1 distribution may differ between resting and activated T cells

  • For TCR internalization studies: Activate T cells with anti-CD3/CD28 antibodies or PMA/ionomycin

  • Consider time-course experiments to capture dynamic changes in FCHO1 localization during T cell activation

  • Include appropriate markers of T cell activation (e.g., CD69, phospho-ZAP70) in multiplexed staining

• Signal amplification techniques:

  • Use tyramide signal amplification to enhance FITC signal without increasing background

  • Consider biotin-streptavidin systems for multi-step amplification

  • For challenging samples, implement rolling circle amplification for antibody detection

• Advanced microscopy approaches:

  • Use deconvolution microscopy to improve signal-to-noise ratio

  • Implement Airyscan or structured illumination microscopy for super-resolution imaging

  • For live T cell imaging, consider lattice light-sheet microscopy to minimize phototoxicity

• T cell-specific counterstaining:

  • Include T cell markers (CD3, CD4, CD8) with spectrally distinct fluorophores

  • Add markers for subcellular compartments of interest (e.g., plasma membrane, endosomes)

  • For clathrin colocalization studies, include clathrin markers compatible with FITC detection

• Flow cytometry optimization:

  • For intracellular FCHO1 detection by flow cytometry, use Triton X-100 or saponin permeabilization

  • Implement stringent gating strategies, including doublet discrimination and viability exclusion

  • Consider using Amnis ImageStream technology to combine flow cytometry with microscopy capabilities

• Patient-derived T cell considerations:

  • When working with T cells from patients with FCHO1 mutations, account for potential lymphopenia

  • Optimize protocols for limited cell numbers (microchamber slides, flow cytometry with acquisition of all events)

  • Include age-matched healthy controls, as T cell characteristics may vary with donor age

• Functional correlation:

  • Correlate FCHO1 staining patterns with functional readouts such as TCR internalization kinetics

  • Implement dual staining for FCHO1 and TCR to visualize their relationship during T cell activation

  • Consider calcium flux assays in parallel to link FCHO1 localization to early T cell activation events

These approaches can help researchers effectively detect and characterize FCHO1 in T cells, facilitating investigations into its role in T cell development, activation, and function, particularly in the context of immunodeficiency disorders associated with FCHO1 mutations .

What controls should be included when studying FCHO1 mutations and their effects on clathrin-mediated endocytosis?

When studying FCHO1 mutations and their effects on clathrin-mediated endocytosis, a comprehensive set of controls is essential to ensure experimental validity and interpretability. Based on published research approaches , the following controls should be included:

• Genetic controls:

  • Wild-type FCHO1-expressing cells as positive controls

  • FCHO1 knockout/knockdown cells as negative controls

  • Cells expressing different FCHO1 mutations (e.g., F-BAR domain mutations like p.A34P versus μHD domain mutations like p.R679P and p.Stop687) to distinguish domain-specific effects

  • Rescue experiments with wild-type FCHO1 to confirm phenotype reversibility

• Protein expression controls:

  • Western blotting to verify expression levels of wild-type and mutant FCHO1 proteins

  • Immunofluorescence to confirm subcellular localization patterns

  • Co-immunoprecipitation to verify disruption of protein-protein interactions (e.g., with EPS15/EPS15R)

  • Assess stability of mutant proteins over time to rule out degradation effects

• Functional endocytosis controls:

  • Cargo-specific controls: Compare TCR internalization with transferrin uptake to distinguish general versus specific endocytic defects

  • Pharmacological controls: Include clathrin inhibitors (e.g., chlorpromazine, Pitstop2) as positive controls for CME disruption

  • Temperature controls: Perform parallel experiments at 37°C (permissive for endocytosis) and 4°C (inhibits endocytosis)

  • Dynamin-dependent control: Use dynamin inhibitors (e.g., Dynasore) to block vesicle scission as a separate control

• Imaging controls:

  • Include markers for plasma membrane, early endosomes, and recycling compartments to track cargo throughout the endocytic pathway

  • Use fluorescently tagged clathrin light chains to directly visualize CCP formation

  • Implement HaloTag or SNAP-tag fusions for pulse-chase analysis of protein dynamics

  • Include untransfected cells within the same field of view as internal controls

• Patient-derived material controls:

  • Age and sex-matched healthy donor cells

  • Cells from patients with different FCHO1 mutations to assess mutation-specific effects

  • Patient cells reconstituted with wild-type FCHO1 as rescue controls

  • Consider including cells from patients with other CME-related defects for comparison

• Domain-specific controls:

  • Express isolated FCHO1 domains (e.g., GFP-FCHO1 (1-609) containing the EFC domain and linker)

  • Create chimeric proteins swapping domains between FCHO1 and related proteins

  • Include point mutations that specifically affect membrane binding, protein interactions, or other functions

• Downstream signaling controls:

  • For T-cell studies, include controls for TCR signaling pathway components (ZAP70, LAT phosphorylation)

  • Control for general cell viability and function independent of endocytosis

  • Include time-course experiments to distinguish primary from secondary effects

By implementing these controls, researchers can confidently attribute observed phenotypes to specific aspects of FCHO1 dysfunction, distinguish between direct and indirect effects, and establish clear mechanistic links between FCHO1 mutations and clathrin-mediated endocytosis defects.