FRL1 Antibody

What is FRL1/FMNL1 and what cellular functions does it regulate?

FRL1 (Formin-like protein 1), also known as FMNL1, is a formin-related protein that plays critical roles in morphogenesis, cytokinesis, and cell polarity. As a member of the conserved formin family expressed across eukaryotes, FRL1 contains characteristic domains including a diaphanous autoregulatory domain (DAD), a formin homology 2 (FH2) domain, and a Rho GTPase-binding/formin homology 3 (GBD/FH3) domain .

FRL1 is primarily located in the cytoplasm and is highly expressed in immune tissues including the spleen, lymph nodes, and bone marrow cells. Its functions are linked to:

• Control of cell motility

• Survival of macrophages

• Cytoskeletal organization

• Podosome formation and dynamics in macrophages

• Cell adhesion processes

Studies have shown that FRL1 is specifically upregulated during monocyte differentiation to macrophages, suggesting developmental regulation of its expression in the myeloid lineage .

What are the key properties of commercially available FRL1 antibodies?

Commercial FRL1 antibodies have specific properties researchers should consider when designing experiments:

The discrepancy between observed (68 kDa) and calculated (121 kDa) molecular weights is notable and should be considered when interpreting Western blot results .

How should FRL1 antibodies be stored and handled for optimal performance?

For optimal performance in research applications, proper storage and handling of FRL1 antibodies is essential:

Store FRL1 antibodies at 4°C for short-term use (up to three months) or at -20°C for long-term storage (stable for up to one year) . Minimize freeze-thaw cycles as repeated cycles can degrade antibody quality and reduce binding efficiency. Most commercial FRL1 antibodies are supplied in PBS containing 0.02% sodium azide as a preservative .

Avoid exposing antibodies to prolonged high temperatures during experimental procedures, as this can lead to denaturation and loss of activity. When working with FRL1 antibodies, maintain cold chain practices during handling, ideally keeping them on ice when in use. For reproducible results, aliquot the antibody into single-use volumes upon receipt to limit freeze-thaw cycles of the stock solution.

How is FRL1 involved in podosome formation and macrophage function?

FRL1 serves as an essential component in podosome biology, particularly in macrophages where podosomes are the sole adhesion structures mediating contact with the extracellular matrix. Research has established that FRL1 localizes specifically to the actin-rich cores of primary macrophage podosomes .

In molecular interaction studies, FRL1 has been shown to co-precipitate with beta-3 integrin but not beta-2 integrin, demonstrating specificity in its integrin associations. Both fixed and live cell fluorescence microscopy confirm that endogenous and overexpressed FRL1 selectively localize to macrophage podosomes, coinciding spatially and temporally with actin and beta-3 integrin in podosome cores .

Functional studies using siRNA-mediated knockdown of FRL1 revealed significant consequences:

These findings support the hypothesis that FRL1 is responsible for modifying actin at the macrophage podosome and plays a crucial role in actin cytoskeleton dynamics during adhesion and migration within tissues .

What experimental approaches are most effective for studying FRL1 function in cellular contexts?

Multiple experimental approaches have proven effective for investigating FRL1 function:

Gene Expression Analysis:
Quantitative RT-PCR combined with Western blotting has successfully demonstrated that FRL1 is specifically upregulated during monocyte differentiation to macrophages (approximately six-fold increase) . This approach is valuable for studying developmental regulation of FRL1.

Protein Interaction Studies:
Co-immunoprecipitation techniques have revealed FRL1's selective association with beta-3 integrin in macrophages . This methodology can be extended to identify other molecular partners of FRL1 in different cellular contexts.

Cellular Localization:
Both fixed and live cell fluorescence microscopy using antibodies against endogenous FRL1 or tagged overexpressed FRL1 have proven effective for tracking its subcellular localization to podosomes . Time-lapse imaging can provide insights into the dynamic recruitment of FRL1 during podosome formation and turnover.

Loss-of-Function Studies:
siRNA-mediated knockdown of FRL1 has successfully demonstrated its functional importance in cell adhesion and podosome dynamics . CRISPR-Cas9 gene editing could provide more complete and stable knockout models for long-term studies.

What are the technical challenges in detecting FRL1 protein expression and how can they be overcome?

Researchers face several technical challenges when detecting FRL1 protein:

Molecular Weight Discrepancy:
A significant challenge is the large difference between the observed molecular weight (68 kDa) and calculated molecular weight (121 kDa) of FRL1 . This discrepancy can lead to misinterpretation of Western blot results.

Methodological solution: Always run appropriate positive controls and molecular weight markers. Consider using multiple antibodies targeting different epitopes of FRL1 to confirm band identity. Additionally, validate antibody specificity using knockdown or knockout samples.

Alternative Splice Variants:
Three alternatively spliced transcript variants of FRL1 have been observed , which can complicate detection and interpretation of results.

Methodological solution: Design primers or use antibodies that can distinguish between different isoforms. Perform RT-PCR to determine which isoforms are expressed in your specific cell type before proceeding with protein studies.

Subcellular Localization:
FRL1's specific localization to podosomes in macrophages may result in signal dilution in whole-cell lysates.

Methodological solution: Consider subcellular fractionation to enrich for cytoskeletal-associated proteins. For microscopy, optimize fixation methods that preserve podosome structures (e.g., using paraformaldehyde rather than methanol fixation).

How can researchers apply FRL1 antibodies to study immune cell function and disease mechanisms?

FRL1 antibodies can be leveraged for multiple research applications in immunology and disease:

Immune Cell Differentiation:
Since FRL1 is upregulated during monocyte-to-macrophage differentiation , antibodies can serve as markers to track this process. Researchers can use FRL1 antibodies in combination with other differentiation markers to characterize macrophage maturation states in various inflammatory conditions.

Cancer Research Applications:
FRL1 has been shown to be upregulated in T cells of patients with non-Hodgkins lymphoma , suggesting potential roles in cancer biology. Immunohistochemistry using FRL1 antibodies could provide insights into abnormal expression patterns in tissue samples from cancer patients.

Podosome Dynamics in Disease:
Podosomes are involved in tissue invasion by inflammatory macrophages and certain transformed cells . FRL1 antibodies can be used to study podosome formation and dynamics in:

• Inflammatory diseases characterized by tissue infiltration

• Cancer cell invasion and metastasis models

• Immune surveillance mechanisms

Therapeutic Target Validation:
As a component of cellular machinery involved in macrophage adhesion and motility, FRL1 could represent a therapeutic target. Antibodies can help validate FRL1 as a target by confirming its expression and localization in disease-relevant tissues.

What emerging technologies might enhance FRL1 antibody development and application?

Recent technological advances offer new opportunities for FRL1 antibody research:

AI-Driven Antibody Design:
RFdiffusion represents a significant advance in using artificial intelligence to generate antibodies. This technology has been fine-tuned to design human-like antibodies by building antibody loops—the intricate, flexible regions responsible for antibody binding . While not specifically developed for FRL1 antibodies, this approach could be adapted to create highly specific antibodies targeting different epitopes or isoforms of FRL1.

The RFdiffusion model produces antibody blueprints that bind user-specified targets, potentially allowing for the creation of antibodies with enhanced specificity or novel functional properties for FRL1 research . This could overcome current limitations in commercial antibody availability.

Single-Cell Analysis:
Integrating FRL1 antibodies into single-cell proteomics workflows could reveal cell-to-cell variability in FRL1 expression and localization, particularly in heterogeneous populations of immune cells.

Live-Cell Imaging Advances:
Super-resolution microscopy techniques combined with minimally disruptive labeling strategies using FRL1 antibody fragments could enable high-resolution tracking of FRL1 dynamics during podosome formation without interfering with normal cellular processes.

Proximity Labeling: Techniques such as BioID or APEX2 proximity labeling could be combined with FRL1 antibodies for immunoprecipitation to identify novel protein interactions within the podosome microenvironment, potentially revealing new functional relationships.