Recombinant Stomatin-3 (sto-3)

What is STOML3 and what are its key functional properties?

STOML3 is a member of the SPFH (stomatin/prohibitin/flotillin/HflK/C) protein superfamily that includes stomatin-like proteins, prohibitins, flotillin/reggie proteins, bacterial HflK/C proteins, and erlins. STOML3 exhibits several key properties:

• It controls membrane mechanics by binding to cholesterol, facilitating force transfer to mechano-gated channels

• It potentiates PIEZO1 and PIEZO2 function by increasing their sensitivity to mechanical stimulations

• It is required for the function of many mechanoreceptors and can modulate mechanotransduction channels and acid-sensing ion channels (ASIC) proteins

• It associates with cholesterol-rich membrane domains (lipid rafts) which appears to be critical for its function

The structure of STOML3 includes domains that are important for cholesterol binding and interaction with membrane proteins, making it a significant modulator of membrane-associated mechanosensation.

How should recombinant STOML3 be expressed for optimal yield and activity?

For effective expression of recombinant STOML3, researchers should consider the following methodological approach:

• Expression System Selection: HEK293T cells have been successfully used for STOML3 expression as evidenced in antibody validation studies . These mammalian cells provide appropriate post-translational modifications essential for STOML3 function.

• Vector Design: Use of eukaryotic expression vectors similar to pEF-Puro.PL3 that has been successfully employed for stomatin family proteins . For fusion proteins, vectors like pEGFP-N3 with appropriate restriction sites (EcoRI/BamHI) can be utilized.

• Expression Enhancement: Consider adding an Alanine linker (Gly-Ala-Ala-Ala) at the C-terminus if creating fusion proteins to maintain proper folding and function .

• Stable vs. Transient Expression: For consistent results, establish stable cell lines through antibiotic selection rather than relying on transient transfection.

What purification strategies yield high-purity recombinant STOML3?

A multi-step purification approach is recommended for obtaining high-purity recombinant STOML3:

• Cell Lysis: Solubilize membranes using 1% Triton X-100 in TNE buffer (20 mM Tris-Cl, pH 8.0, 130 mM NaCl, 5 mM EDTA) with protease inhibitors (10 μg/ml aprotinin and leupeptin, 1 μg/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride) at room temperature for 10 minutes .

• Density Gradient Centrifugation: Apply solubilized proteins to a linear sucrose density gradient (15–50%) and centrifuge at high speed (e.g., 40,000 rpm at 4°C for 19 hours) .

• Immunoprecipitation: For tagged proteins, use appropriate antibodies or anti-tag affinity resins. For native STOML3, specific antibodies like those used for stomatin (GARP-50) have proven effective .

• Quality Control: Verify purification by SDS-PAGE and Western blotting using validated antibodies such as ab254769 that has been tested for specificity with human STOML3 .

The purity and activity of the final product should be confirmed through functional assays measuring its interaction with known binding partners or effects on mechanosensitive channels.

How can the functional activity of recombinant STOML3 be verified?

To confirm that purified recombinant STOML3 retains its functional activity, researchers should employ multiple complementary approaches:

• Cholesterol Binding Assay: Utilize [³H]photocholesterol cross-linking methodology as described for stomatin proteins. Incubate cells expressing STOML3 with radiolabeled photocholesterol, perform UV crosslinking, immunoprecipitate STOML3, and analyze by SDS-PAGE and autoradiography .

• Membrane Association Analysis: Examine the association with detergent-resistant membranes (DRMs) through sucrose gradient centrifugation after Triton X-100 solubilization, which indicates proper incorporation into cholesterol-rich domains .

• Piezo Channel Modulation: Conduct patch-clamp electrophysiology to measure STOML3's ability to potentiate PIEZO1 and PIEZO2 currents in response to mechanical stimulation. Functional STOML3 should increase channel sensitivity to mechanical forces .

• Oligomerization Assessment: Analyze the formation of homo-oligomers through techniques such as blue native PAGE or chemical crosslinking followed by SDS-PAGE, as oligomerization is critical for stomatin family protein function .

• Proximity Ligation Assays (PLA): Perform in situ PLA to verify protein-protein interactions between STOML3 and its known binding partners, similar to methods used to demonstrate stomatin-AE1 interactions .

What are the optimal storage conditions for maintaining STOML3 stability?

To maintain the stability and functional integrity of recombinant STOML3, researchers should consider the following storage parameters:

• Temperature: Store purified protein at -80°C for long-term storage. For short-term use (1-2 weeks), 4°C storage may be suitable if the protein is in an appropriate stabilizing buffer.

• Buffer Composition: A recommended storage buffer includes:

  • 20 mM Tris-HCl, pH 7.4-8.0

  • 150 mM NaCl

  • 5% glycerol (as cryoprotectant)

  • 0.5-1 mM DTT or 2-5 mM β-mercaptoethanol (to prevent oxidation)

• Avoiding Freeze-Thaw Cycles: Prepare single-use aliquots to minimize freeze-thaw cycles which can cause protein denaturation and aggregation.

• Protein Concentration: Maintain concentrations above 0.1 mg/ml to prevent adsorption to container surfaces, but below levels that might promote aggregation (typically <5 mg/ml).

• Additives: Consider adding stabilizers such as 0.1% cholesterol-rich liposomes or nanodiscs to maintain the native conformation of STOML3, as its function is closely associated with cholesterol binding .

How does STOML3 mechanistically regulate Piezo channel sensitivity?

The molecular mechanism by which STOML3 regulates Piezo channels involves both direct protein-protein interactions and indirect effects on membrane properties:

• Membrane Mechanics Modulation: STOML3 controls membrane mechanics by binding cholesterol, thereby creating a more favorable mechanical environment for force transfer to mechanosensitive channels . This membrane stiffening effect is crucial as it:

  • Enhances the efficiency of force transmission to embedded channels

  • Reduces the force threshold required for channel activation

  • Creates localized membrane domains with altered mechanical properties

• Direct Channel Interaction: STOML3 appears to interact directly with mechanosensitive channels, potentially stabilizing specific conformational states that favor channel opening at lower mechanical forces. This interaction is thought to involve the conserved PHB/SPFH domain of STOML3 .

• Experimental Approaches to Study This Mechanism:

  • Use atomic force microscopy (AFM) to measure changes in membrane stiffness in the presence and absence of STOML3

  • Employ FRET-based assays to detect conformational changes in Piezo channels when interacting with STOML3

  • Design chimeric proteins between STOML3 and other stomatin family members to identify domains critical for Piezo sensitization

  • Perform site-directed mutagenesis targeting the CRAC/CARC and ORA/CARC domains (residues 55-68 and 263-273) that are important for cholesterol binding

The dual action of STOML3—binding cholesterol to alter membrane properties while also directly interacting with channel proteins—represents a sophisticated mechanism for fine-tuning cellular mechanosensitivity.

What structural mutations in recombinant STOML3 provide insights into function?

Strategic mutations in STOML3 can reveal critical structure-function relationships relevant to its role in mechanosensation:

To implement these mutations effectively:

• Use PCR-based site-directed mutagenesis with mutagenic oligonucleotides containing the desired mutations .

• Verify mutations through DNA sequencing before expression.

• Express both wild-type and mutant proteins in parallel to enable direct functional comparisons.

• Assess the impact of mutations through multiple assays including:

  • Cholesterol binding using [³H]photocholesterol cross-linking

  • Membrane domain association via sucrose gradient fractionation of detergent-resistant membranes

  • Oligomerization status using blue native PAGE or crosslinking approaches

  • Functional effects on Piezo channel sensitivity using electrophysiology

  • Subcellular localization and dynamics using confocal microscopy

This comprehensive mutational analysis approach has successfully revealed functional domains in stomatin family proteins and can be applied to understand STOML3's specific roles in mechanosensation.

How can researchers effectively study STOML3's role in lipid raft dynamics?

Investigating STOML3's function in lipid raft dynamics requires specialized techniques that preserve native membrane organization:

• Detergent-Resistant Membrane Isolation:

  • Solubilize cell membranes with 1% Triton X-100 in TNE buffer at room temperature

  • Separate fractions using 15-50% sucrose density gradient ultracentrifugation

  • Collect fractions and analyze by Western blotting for STOML3 and lipid raft markers

• Cholesterol Depletion Studies:

  • Treat cells with methyl-β-cyclodextrin (MβCD) to deplete membrane cholesterol

  • Compare STOML3 localization and function before and after treatment

  • Examine whether cholesterol depletion and STOML3 deficiency show interdependent effects on mechanosensitivity

• Super-Resolution Microscopy:

  • Employ techniques such as STORM, PALM, or STED microscopy to visualize nanoscale organization of STOML3 within membrane domains

  • Use dual-color imaging to examine colocalization with other raft components

  • Implement live-cell imaging to track dynamic association with rafts

• Fluorescence Recovery After Photobleaching (FRAP):

  • Measure lateral mobility of fluorescently tagged STOML3 in different membrane regions

  • Compare diffusion rates in wild-type cells versus those with altered cholesterol content

  • Analyze the impact of cytoskeletal disruption on STOML3 mobility, particularly focusing on the C-terminal region (residues 264-288) that interacts with the actin cytoskeleton

• Proximity Ligation Assays:

  • Use in situ PLA to detect and quantify interactions between STOML3 and other raft-associated proteins

  • Apply this technique in both cell lines and primary neurons to confirm physiological relevance

• Lipidomic Analysis:

  • Perform mass spectrometry-based lipidomics on STOML3-containing membrane fractions

  • Identify specific lipid species that preferentially associate with STOML3

  • Compare lipid compositions in wild-type versus STOML3-deficient samples

This multi-faceted approach provides comprehensive insights into how STOML3 organizes and functions within specialized membrane domains to regulate mechanosensation.

What experimental paradigms best elucidate STOML3's role in sensory neuron mechanotransduction?

To comprehensively investigate STOML3's function in sensory neuron mechanotransduction, researchers should implement multiple complementary approaches:

• Electrophysiological Characterization:

  • Perform patch-clamp recordings on dorsal root ganglion (DRG) neurons from wild-type and STOML3-deficient mice

  • Apply calibrated mechanical stimuli using piezo-driven probes or pressure clamps

  • Analyze mechanically-activated current properties including threshold, amplitude, kinetics, and adaptation

  • Reconstitute the system by expressing recombinant STOML3 in STOML3-deficient neurons to verify rescue of function

• Ex Vivo Skin-Nerve Preparations:

  • Isolate skin with attached nerves from wild-type and STOML3-deficient mice

  • Record from identified mechanoreceptor types while applying precisely controlled mechanical stimulation

  • Quantify differences in firing thresholds, frequency response, and adaptation properties

  • Test the effects of acute cholesterol depletion on mechanoreceptor function in both genotypes

• Calcium Imaging of Mechanosensitive Responses:

  • Culture DRG neurons from wild-type and STOML3-deficient mice

  • Load cells with calcium-sensitive dyes or express genetically-encoded calcium indicators

  • Apply mechanical stimuli while monitoring calcium transients

  • Analyze population responses to identify subpopulations of mechanosensitive neurons affected by STOML3 deficiency

• In Vivo Behavioral Testing:

  • Perform tactile sensitivity assays (von Frey, dynamic touch, texture discrimination) on wild-type and STOML3-deficient mice

  • Assess mechanical pain thresholds and allodynia development after inflammation or nerve injury

  • Test the effects of cholesterol depletion on tactile allodynia in both genotypes

  • Compare responses before and after specific pharmacological manipulation of STOML3-dependent pathways

• Single-Cell Transcriptomics and Proteomics:

  • Perform single-cell RNA sequencing on sensory neurons to identify STOML3-dependent gene expression programs

  • Correlate STOML3 expression levels with specific mechanoreceptor subtypes

  • Analyze changes in the expression of mechanotransduction components in STOML3-deficient neurons

This multidisciplinary approach allows for correlation between molecular interactions, cellular responses, and behavioral outcomes, providing a comprehensive understanding of STOML3's role in mechanosensation.

How can researchers measure STOML3-mediated changes in membrane mechanics?

Quantifying STOML3's effects on membrane mechanical properties requires specialized biophysical techniques:

• Atomic Force Microscopy (AFM):

  • Prepare cell membrane patches or reconstituted proteoliposomes with and without STOML3

  • Use AFM in force spectroscopy mode to measure local membrane stiffness

  • Compare Young's modulus values between STOML3-containing and STOML3-deficient membranes

  • Correlate stiffness changes with STOML3 concentration and cholesterol content

• Membrane Tether Pulling:

  • Use optical tweezers to pull membrane tethers from cells expressing or lacking STOML3

  • Measure the force required for tether formation and extension

  • Analyze tether radius and membrane tension as indicators of membrane mechanical properties

  • Compare results before and after cholesterol depletion to determine interdependence

• Fluorescence-Based Membrane Tension Sensors:

  • Express genetically-encoded tension sensors (such as FliptR or PicoYellow) in cells

  • Monitor changes in fluorescence lifetime or intensity that correlate with membrane tension

  • Compare tension values in wild-type cells versus those overexpressing or lacking STOML3

  • Perform real-time measurements during mechanical stimulation to assess dynamic changes

• Micropipette Aspiration:

  • Apply controlled suction pressure to cell membranes through a micropipette

  • Measure membrane deformation as a function of applied pressure

  • Calculate membrane elastic modulus from the pressure-deformation relationship

  • Compare properties between cells with different STOML3 expression levels

• Molecular Dynamics Simulations:

  • Create computational models of membrane patches containing STOML3 and cholesterol

  • Simulate mechanical deformation of the membrane under various forces

  • Analyze how STOML3-cholesterol interactions affect membrane response to mechanical perturbation

  • Test predictions from simulations with experimental approaches

These techniques provide quantitative measurements of how STOML3 modifies membrane mechanical properties, which is critical for understanding its role in facilitating mechanotransduction through Piezo and other mechanosensitive channels.

What are the comparative functional differences between STOML3 and other stomatin family proteins?

The stomatin protein family includes several members with distinct but related functions. Understanding their comparative properties provides valuable insights:

To experimentally investigate these differences:

• Domain Swapping Experiments:

  • Create chimeric proteins exchanging domains between STOML3 and other family members

  • Express these chimeras in appropriate cell systems

  • Test their ability to modulate Piezo channels, bind cholesterol, and alter membrane properties

  • Identify critical regions that determine the unique functions of STOML3

• Comparative Binding Studies:

  • Perform pull-down assays with recombinant stomatin family proteins

  • Identify differential protein binding partners through mass spectrometry

  • Compare cholesterol binding efficiency using [³H]photocholesterol cross-linking

  • Analyze relative affinities for specific lipid compositions

• Cross-Complementation Assays:

  • Express each stomatin family protein in STOML3-deficient sensory neurons

  • Test ability to rescue mechanosensitivity defects

  • Quantify differences in mechanically-activated current properties

  • Correlate functional rescue with specific molecular features

This comparative approach reveals how evolutionary diversification of the stomatin family has led to specialized functions while maintaining core structural features, providing insights for both basic research and potential therapeutic targeting.