TMOD3 Antibody

What is TMOD3 and why is it important in cellular biology?

TMOD3 (tropomodulin 3) is a widely expressed actin-binding protein that caps the pointed ends of actin filaments, preventing both elongation and depolymerization. The TMOD3/tropomyosin complex contributes to the formation of short actin protofilaments that define membrane skeleton geometry . TMOD3 plays critical roles in regulating actin dynamics throughout the cell, particularly in processes including cell migration, lamellipodial formation, and cytoskeletal organization . Its importance is highlighted in vascular endothelial cells, where modulating TMOD3 levels directly affects cell motility—with overexpression decreasing migration and depletion accelerating it . Additionally, TMOD3 has been implicated in megakaryocyte development and platelet formation .

How does TMOD3 differ functionally from other tropomodulin isoforms?

While all tropomodulins (Tmods) share the ability to cap actin filament pointed ends, TMOD3 exhibits a ubiquitous expression pattern, unlike the tissue-specific distributions of TMOD1 (erythrocytes, striated muscle, and some neurons), TMOD2 (neuronal), and TMOD4 (skeletal muscle) . Functionally, TMOD3 uniquely localizes to both contractile and non-contractile actin bundles, whereas TMOD1 appears restricted to contractile actin bundles only . TMOD3 shows distinctive subcellular localization, with a portion enriched in leading edge ruffles and lamellipodia . Furthermore, unlike other isoforms, TMOD3 can be phosphorylated at Ser25 by AMPK, mediating GLUT4 translocation and glucose uptake .

What are the molecular characteristics of TMOD3 relevant to antibody selection?

TMOD3 is a 40 kDa protein consisting of 352 amino acids . Its molecular structure includes two actin-binding sites: an N-terminal tropomyosin-dependent site and a C-terminal tropomyosin-independent site containing leucine-rich repeats (LRRs) . When selecting antibodies, researchers should consider that TMOD3 has high sequence homology with other Tmod family members, particularly in the C-terminal domain, which can affect antibody specificity . The protein is predominantly cytoplasmic but associates with the cytoskeleton, with approximately 30-40% of endogenous TMOD3 being cytoskeleton-associated in cellular fractionation studies .

What are the validated applications for TMOD3 antibodies and their recommended protocols?

TMOD3 antibodies have been validated for multiple applications:

Western Blot (WB):

Immunoprecipitation (IP):

Immunohistochemistry (IHC):

ELISA:

How can I quantify endogenous TMOD3 levels in experimental cell lines?

To quantify endogenous TMOD3 levels, implement quantitative immunoblotting against known standards of recombinant TMOD3 protein. For example, in HMEC-1 cells, researchers loaded cell lysate from known numbers of cells alongside standards of purified recombinant human TMOD3 on the same gel . Calculate the total intracellular TMOD3 concentration by determining the mean cell volume (from mean cell diameter measurements of trypsinized cells) and compare with the standard curve. In HMEC-1 cells, this approach yielded an estimated total TMOD3 concentration of approximately 0.5 μM . For more precise quantification in distinct cellular fractions, perform Triton X-100 extraction to separate cytoskeleton-associated TMOD3 (~30-40% of total) from soluble pools .

What is the optimal approach for detecting TMOD3 phosphorylation in experimental systems?

For detecting TMOD3 phosphorylation, particularly at sites like Ser25 that are regulated by AMPK, a multi-technique approach is recommended:

• Immunoprecipitation followed by LC-MS/MS analysis:

  • Express FLAG-tagged TMOD3 in cells (e.g., HEK293T) with relevant kinases

  • Purify using anti-FLAG M2 Affinity gel

  • Separate proteins by SDS-PAGE (12% precast gels)

  • Perform in-gel digestion with trypsin after reduction and alkylation

  • Analyze peptides by LC-MS/MS to identify phosphorylation sites

• In vitro kinase assays:

  • Incubate 1 μg purified FLAG-TMOD3 with 1 μg recombinant kinase (e.g., AMPK)

  • Use kinase buffer containing 0.2 mM ATP at 30°C for 30 minutes

  • Detect phosphorylation using anti-phospho-serine antibodies

  • For more sensitive detection, incorporate [32P]ATP and perform autoradiography

• Phospho-specific antibodies:

  • Generate or obtain antibodies that specifically recognize phosphorylated TMOD3 at the site of interest

  • Validate specificity using phosphatase treatment controls

How can I effectively analyze TMOD3's role in actin dynamics and cell migration?

To analyze TMOD3's impact on actin dynamics and cell migration, implement a comprehensive approach:

• Modulation of TMOD3 levels:

  • Overexpression: Use adenovirus-mediated GFP-TMOD3 expression for consistent overexpression in nearly all cells

  • Knockdown: Apply RNA interference techniques

  • Knockout: Generate TMOD3-deficient cells using CRISPR/Cas9

• Free pointed end quantification:

  • Extract monomeric actin with detergent before fixation

  • Stain with fluorescently labeled DNase I, which binds to G-actin and free pointed ends

  • Quantify DNase I staining intensity, excluding regions of nuclear staining

• Migration assays:

  • Wound healing (scratch) assays

  • Transwell migration assays

  • Live-cell imaging to track single-cell motility

• Actin structure analysis:

  • Phalloidin staining to visualize F-actin

  • Tropomyosin staining to identify stable actin filaments

  • Arp2/3 and cortactin staining to visualize lamellipodial structures

• Quantitative analysis:

  • Measure lamellipodial width and protrusion rates

  • Quantify relative levels of F-actin, free barbed ends, and Arp2/3 complex

  • Perform biochemical fractionation to determine cytoskeleton-associated versus soluble TMOD3 pools

What techniques are most effective for studying TMOD3-tropomyosin interactions in cells?

For investigating TMOD3-tropomyosin interactions in cellular contexts:

• Proximity Ligation Assay (PLA):

  • Express tagged TMOD3 (e.g., ClFP-tagged) in cells

  • Fix cells and perform PLA between exogenous TMOD3 and endogenous tropomyosin isoforms

  • Count PLA reaction puncta in different cellular compartments (cell bodies, processes, etc.)

  • This technique allows detection of proteins within 30-40 nm of each other

• Co-immunoprecipitation:

  • Lyse cells in conditions that preserve protein-protein interactions

  • Immunoprecipitate TMOD3 and probe for associated tropomyosin isoforms

  • Alternatively, immunoprecipitate tropomyosin and detect TMOD3

• Biochemical fractionation and western blotting:

  • Deplete TMOD3 through knockdown or knockout

  • Analyze changes in tropomyosin isoform levels by western blotting

  • As shown in research, depletion of both TMOD3 and TMOD1 drastically reduces levels of all tropomyosin isoforms

• Live cell imaging with fluorescently-tagged proteins:

  • Co-express fluorescently-tagged TMOD3 and tropomyosin

  • Perform FRET analysis to detect direct interactions

  • Track co-localization during dynamic cellular processes

What experimental system is best for investigating TMOD3's role in platelet formation?

Based on research findings, the most appropriate experimental system for studying TMOD3's role in platelet biogenesis is:

• Mouse fetal liver-derived megakaryocytes (MKs):

  • Tmod3 knockout embryos (Tmod3^-/-^) provide an excellent model, as they show hemorrhaging at E14.5 with platelet abnormalities

  • Isolate fetal liver cells at E13.5-E14.5 and culture them in the presence of thrombopoietin to promote MK differentiation

• Key analyses to perform:

  • MK differentiation assessment: Flow cytometry for ploidy analysis and MK-specific markers

  • Demarcation membrane system (DMS) analysis: Transmission electron microscopy to assess DMS formation

  • Proplatelet formation assay: Culture MKs on fibrinogen-coated surfaces and quantify proplatelet extension

  • Granule distribution: Immunofluorescence for von Willebrand factor (VWF) to assess granule content and distribution

  • F-actin organization: Phalloidin staining to visualize F-actin structures in MKs and proplatelet buds

  • Platelet analysis: Count and measure platelet size using flow cytometry and assess GPIbα distribution via confocal microscopy

This model is particularly valuable because TMOD3 knockout causes embryonic lethality by E18.5, but fetal liver-derived MKs can be studied before this timepoint.

How can I address non-specific nuclear staining when using TMOD3 antibodies for immunofluorescence?

• Validation strategies:

  • Always include a TMOD3 knockout or knockdown control to identify non-specific staining

  • Test multiple antibodies from different sources and compare staining patterns

  • Perform peptide competition assays to confirm specificity

• Technical optimizations:

  • Increase blocking duration and concentration (use 5-10% normal serum from the species of secondary antibody)

  • Include 0.1-0.3% Triton X-100 in blocking and antibody solutions

  • Try different fixation methods (paraformaldehyde vs. methanol)

  • Implement a pre-adsorption step by incubating the antibody with cell/tissue lysate from TMOD3 knockout samples

• Analysis considerations:

  • When quantifying TMOD3 signals, exclude nuclear regions from analysis

  • Focus on established subcellular localizations like stress fibers, lamellipodia, and cytoskeletal structures

What are the critical parameters for optimizing western blot detection of TMOD3?

For optimal western blot detection of TMOD3:

Additionally, for quantitative western blotting, always include a housekeeping protein control, prepare freshly made buffers, and ensure even protein transfer by staining membranes with Ponceau S prior to blocking.

How can I distinguish between TMOD3 and other tropomodulin isoforms in my experimental system?

Distinguishing between TMOD isoforms is crucial due to their structural similarities. Use these strategies:

• Antibody selection and validation:

  • Choose isoform-specific antibodies and validate them against recombinant proteins of all TMOD isoforms

  • For example, some polyclonal TMOD3 antibodies cross-react with TMOD1, requiring additional controls

  • Use monoclonal antibodies when available, as they typically offer higher specificity

• Molecular weight differentiation:

  • TMOD3: 40 kDa

  • TMOD1: 43 kDa

  • TMOD2: 41 kDa

  • TMOD4: 39 kDa
    Use high-resolution gels (10-12%) to separate these closely sized proteins

• Expression pattern analysis:

  • TMOD1: Primarily in erythrocytes, cardiac and skeletal muscle

  • TMOD2: Predominantly neuronal

  • TMOD3: Ubiquitous

  • TMOD4: Skeletal muscle-specific
    This tissue specificity can help inform expectations

• Gene-specific approaches:

  • Design PCR primers or siRNAs that target unique regions of each TMOD isoform

  • Verify knockdown specificity by testing effects on all TMOD isoforms

• Subcellular localization patterns:

  • TMOD3 localizes to both contractile and non-contractile actin bundles and is partially present in lamellipodia

  • TMOD1 is typically restricted to contractile actin bundles
    This differential localization can help distinguish isoforms in imaging studies

How do mutations in TMOD3's actin-binding sites affect its function in cellular contexts?

TMOD3 contains two actin-binding sites (ABS): an N-terminal tropomyosin-dependent site (ABS1) and a C-terminal tropomyosin-independent site (ABS2) containing leucine-rich repeats . Research on tropomodulins reveals:

What is the relationship between TMOD3 phosphorylation and its role in glucose uptake?

TMOD3 phosphorylation, particularly at Ser25, plays a critical role in regulating GLUT4 plasma membrane insertion and glucose uptake:

• Phosphorylation mechanism:

  • AMPK (AMP-activated protein kinase) directly phosphorylates TMOD3 at Ser25

  • This phosphorylation can be detected using:

    • LC-MS/MS analysis of purified FLAG-TMOD3 co-expressed with active AMPK

    • In vitro kinase assays with recombinant AMPK and FLAG-TMOD3

    • Anti-phospho-serine antibodies

• Functional significance:

  • TMOD3 serves as a key AMPK effector in muscle cells

  • Phosphorylation of TMOD3 mediates GLUT4 insertion into the plasma membrane

  • This process is essential for insulin-stimulated glucose uptake

• Experimental approaches:

  • Generate phospho-mimetic (S25D) and phospho-deficient (S25A) TMOD3 mutants

  • Express these mutants in cells and assess:

    • GLUT4 translocation using GLUT4-GFP fusion proteins

    • Glucose uptake via radioactive glucose analogs

    • Changes in actin dynamics that might affect vesicle trafficking

    • Co-localization with components of the GLUT4 trafficking machinery

• Relationship to actin dynamics:

  • TMOD3's actin-capping function may be modulated by phosphorylation

  • Investigate how phosphorylation affects:

    • TMOD3's association with the cytoskeleton

    • Its interaction with tropomyosin

    • Local remodeling of actin to facilitate vesicle fusion

How can I design experiments to investigate the interplay between TMOD3 and the Arp2/3 complex in lamellipodia formation?

To investigate the relationship between TMOD3 and the Arp2/3 complex in lamellipodia:

• Rationale for investigation:

  • TMOD3 depletion results in more pronounced lamellipodia-like protrusions with increased Arp2/3 complex

  • TMOD3 overexpression decreases cell motility and affects free barbed ends and Arp2/3 complex in lamellipodia

• Experimental design approaches:

  a. Manipulate TMOD3 levels and assess Arp2/3 activity:

  • Use TMOD3 knockout/knockdown and overexpression systems

  • Quantify Arp2/3 complex levels and distribution by immunofluorescence

  • Measure lamellipodial width and dynamics

  • Analyze active Arp2/3 using phospho-specific antibodies against activated Arp2

  b. Perform reciprocal experiments with Arp2/3 inhibition:

  • Use small molecule Arp2/3 inhibitors (CK-666) or siRNA against Arp2/3 components

  • Determine how Arp2/3 inhibition affects TMOD3 localization and function

  • Assess whether Arp2/3 inhibition can rescue phenotypes caused by TMOD3 depletion

  c. Investigate actin filament populations:

  • Use super-resolution microscopy to distinguish Arp2/3-nucleated filaments from tropomyosin-decorated filaments

  • Perform live-cell imaging with fluorescently tagged TMOD3 and Arp2/3 components

  • Quantify the relative abundance and distribution of different actin populations

  d. Examine tropomyosin involvement:

  • TMOD3 depletion affects tropomyosin levels, which may indirectly influence Arp2/3 function

  • Test whether tropomyosin overexpression can rescue effects of TMOD3 depletion

  • Investigate competition between tropomyosin and Arp2/3 for actin filaments

• Advanced analysis techniques:

  • Fluorescence recovery after photobleaching (FRAP) to measure turnover rates of actin in lamellipodia

  • Proximity ligation assays to detect potential interactions between TMOD3 and Arp2/3 complex components

  • Correlative light and electron microscopy to examine detailed actin network architecture

These experiments would provide insights into how TMOD3-mediated pointed-end capping influences Arp2/3-dependent actin networks and how these two actin-regulatory systems interact to control lamellipodial dynamics and cell migration.