Recombinant Rat Protein FAM101B (Fam101b)

What is FAM101B/RefilinB and what is its molecular profile in rat models?

FAM101B (Family with sequence similarity 101, member B), also known as RefilinB (RFLNB) or CFM1, is a hydrophilic protein enriched in proline that functions as an actin regulator . In rats, FAM101B has the following characteristics:

The protein has a secondary structure primarily composed of β structures and coiled domains lacking α helices . It contains a conserved N-terminal sequence with a DSG(X)2–4S motif that mediates degradation of short-lived proteins, similar to motifs found in nuclear transcription factors involved in TGF-β-dependent EMT signaling .

How is rat FAM101B structurally and functionally related to its human and mouse orthologs?

Rat FAM101B shares significant homology with human and mouse orthologs, reflecting evolutionary conservation of this protein family:

Both human and rat FAM101B contain the characteristic N-terminal motif and function as part of the refilin family of actin regulators that are widely expressed during early embryonic development . The high degree of conservation suggests critical biological functions that have been maintained across mammalian species .

What is the expression pattern and tissue distribution of FAM101B in rat models?

FAM101B shows specific expression patterns in rat tissues:

• Widely expressed during early embryonic development

• Shows variable expression levels across different brain regions according to Allen Brain Atlas data

• In fibroblasts, shows heterogeneous expression with highest levels in cells demonstrating apical FLNA staining

• Expression is upregulated during specific cell differentiation events, particularly during epithelial-mesenchymal transition (EMT) mediated by TGF-β

What is the cellular function of FAM101B and its interaction with the actin cytoskeleton?

FAM101B (RefilinB) plays a crucial role in organizing perinuclear actin networks:

• Converts Filamin A (FLNA) from an actin branching protein into an actin bundling protein

• In NIH 3T3 fibroblasts, the RefilinB/FLNA complex organizes perinuclear actin filament bundles into a structure called the actin cap

• Controls formation of perinuclear actin networks that accompany nuclear shape changes during epithelial-mesenchymal transition

• Affects nuclear height - cells with high RefilinB expression show reduced nuclear height compared to control cells

• RefilinB-organized perinuclear actin filaments are immunostained with myosin II antibody, indicating they are contractile actin bundles

The RefilinB/FLNA complex is essential for maintaining nuclear shape and position. When RefilinB is downregulated via shRNA in NIH 3T3 cells, perinuclear actin bundles disappear, the actin cap organization is disrupted, and nuclear height increases .

How does FAM101B specifically interact with Filamin A (FLNA) at the molecular level?

The interaction between FAM101B and FLNA involves specific binding domains and induces functional transformation:

Studies using fluorescence microscopy and electron microscopy have revealed that FLNA alone forms homogenous actin filament networks, but in the presence of RefilinB, it forms large F-actin bundles even at a low ratio of FLNA to actin (1:100) .

What is the role of FAM101B during epithelial-mesenchymal transition (EMT)?

FAM101B plays a specific role during EMT:

• RefilinB mRNA is upregulated during EMT mediated by TGF-β

• In epithelial normal murine mammary gland (NmuMG) cells, the RefilinB/FLNA complex controls formation of a new perinuclear actin network that accompanies nuclear shape changes during EMT

• RefilinB contains a motif also found in nuclear transcription factors involved in TGF-β-dependent EMT signaling

• This involvement in EMT suggests a potential role in tissue differentiation and organ development, as EMT is a biological process crucial for the differentiation of multiple tissues and organs

What are the recommended protocols for reconstituting and handling recombinant rat FAM101B?

Based on standard protocols for similar recombinant proteins:

When working with His-tagged versions of the protein, the following specific handling guidelines apply:

• For co-immunoprecipitation studies, maintain appropriate buffer conditions to preserve protein interactions

• For structural studies, consider the impact of the tag on protein folding and interactions

What are the optimal storage conditions for maintaining recombinant rat FAM101B stability?

Proper storage is critical for maintaining protein activity:

For lyophilized preparations:

• After reconstitution, aliquot to minimize freeze-thaw cycles

• Use a manual defrost freezer for storage

• Consider adding carrier proteins (such as BSA) if diluting to low concentrations for long-term storage

What expression systems and purification methods are most effective for producing functional recombinant rat FAM101B?

Several expression systems have been used successfully:

Purification approaches:

• Affinity chromatography using His-tag is common, with purity typically >80%

• For higher purity (>90%), additional chromatography steps may be necessary

• When studying interactions with FLNA, co-expression and co-purification might be advantageous

For functional studies, His-EGFP-tagged FAM101B has been successfully used to visualize actin bundling in vitro .

How can recombinant rat FAM101B be utilized to study perinuclear actin dynamics?

Recombinant FAM101B is a valuable tool for investigating perinuclear actin organization:

• In vitro actin bundling assays: Mixing recombinant FAM101B with FLNA and actin can demonstrate direct effects on actin organization, viewable by fluorescence microscopy after costaining F-actin with fluorescent phalloidin or by electron microscopy

• Live cell imaging: Expressing RefilinB-GFP constructs allows visualization of actin fiber reorganization in real-time, showing formation of parallel actin filament bundles organized into a crescentic shell above the nucleus

• Nuclear height measurement: Quantitative comparison of nuclear height in cells expressing recombinant FAM101B versus controls can demonstrate functional effects on nuclear morphology

• Knockdown-rescue experiments: Depleting endogenous FAM101B with shRNA and reintroducing recombinant protein allows structure-function analysis to identify critical domains

These approaches can help delineate the molecular mechanisms by which FAM101B regulates the perinuclear actin cytoskeleton and nuclear architecture.

What are the current research challenges in studying FAM101B-FLNA interactions?

Researchers face several challenges when investigating FAM101B-FLNA interactions:

• Complex binding mechanism: The presence of multiple binding domains complicates the mapping of precise interaction interfaces. While FLNA repeat 21 is critical, deletion of this domain alone is not sufficient to inhibit interaction

• Structural determination: The hydrophilic nature of FAM101B with its predominant β structures and coiled domains makes crystallographic studies challenging

• Temporal dynamics: As FAM101B is a short-lived protein containing a degradation motif, studying its dynamics in real time requires sophisticated live imaging approaches

• Physiological relevance: Connecting in vitro biochemical interactions to in vivo cellular phenotypes, particularly during development and disease states, remains challenging

• Redundancy with RefilinA: The presence of the related family member RefilinA (FAM101A) may provide functional redundancy, complicating loss-of-function studies

Innovative approaches combining structural biology, real-time imaging, and physiological models are needed to address these challenges.

What methods can be used to investigate FAM101B phosphorylation and other post-translational modifications?

Post-translational modifications of FAM101B can be investigated using various approaches:

Given that FAM101B contains motifs found in TGF-β-regulated transcription factors, particular attention should be paid to modifications that might occur in response to TGF-β signaling during EMT .

What are the implications of FAM101B in developmental disorders related to FLNA mutations?

FAM101B research has potential implications for understanding FLNA-related disorders:

• FLNA mutations cause a wide range of developmental malformations in the heart, skeleton, and brain

• Since FAM101B converts FLNA from an actin branching protein to a bundling protein, dysregulation of this interaction might contribute to disease phenotypes

• Abnormal perinuclear actin dynamics due to disrupted FAM101B-FLNA interactions could contribute to neuronal migration defects seen in patients with FLNA mutations

• The role of FAM101B in EMT suggests its potential involvement in developmental processes that are disrupted in FLNA-associated conditions

Understanding how FAM101B regulates FLNA function could provide insights into the molecular mechanisms underlying the broad spectrum of diseases caused by different FLNA mutations.

How might FAM101B function as a potential therapeutic target in cytoskeletal disorders?

While still largely theoretical, FAM101B could represent a novel therapeutic target:

• Modulating nuclear mechanics: As FAM101B regulates perinuclear actin organization and nuclear height, targeting this pathway might allow manipulation of nuclear mechanics in disease states characterized by abnormal nuclear morphology

• EMT regulation: Given its role in EMT, modulating FAM101B activity could potentially influence developmental and pathological processes involving EMT, such as tissue fibrosis or cancer metastasis

• FLNA function rescue: In cases where FLNA mutations disrupt its bundling capacity, enhancing FAM101B function might potentially compensate by reinforcing the remaining bundling activity

• Neuronal migration: As FAM101B may influence neuronal migration through perinuclear actin dynamics, it could represent a target for developmental neurological disorders

Research is still at an early stage, and significant validation in disease models would be required before therapeutic applications could be considered.

What experimental models are best suited for studying FAM101B function in development and disease?

Several experimental models offer advantages for FAM101B research:

When selecting an experimental model, researchers should consider the specific aspect of FAM101B biology they wish to investigate, as different models highlight different functional aspects of this versatile protein.