Recombinant 71 kDa F-actin-binding protein

What is the 71 kDa F-actin-binding protein Abp140 and how does it function in live cell imaging?

Abp140 is a 71 kDa actin filament crosslinking protein featuring a short N-terminal actin-binding sequence and a C-terminal tRNA methyltransferase domain. For over two decades, endogenously-tagged Abp140-GFP has served as the primary tool for visualizing actin cables in live yeast cells . The protein effectively decorates dynamic actin structures, enabling researchers to observe cytoskeletal organization and movements in real time.

The methodology for utilizing Abp140 in live imaging typically involves:

• Endogenous tagging with fluorescent proteins (typically GFP or mNeonGreen)

• Live cell confocal or widefield fluorescence microscopy

• Time-lapse imaging to capture dynamic changes in actin organization

• Analysis of cable movement, assembly, and disassembly rates

Interestingly, Abp140 demonstrates compartmentalized decoration patterns, with stronger signals in mother cell actin cables compared to bud compartment cables . This asymmetric decoration is F-actin binding dependent but does not rely on which formin (Bni1 or Bnr1) assembles the cables .

How does Abp140 compare with other F-actin binding proteins in experimental systems?

Several F-actin binding proteins serve as research tools, each with distinct properties that influence their experimental applications. The table below summarizes key differences:

Unlike cofilin, which functions primarily to sever actin filaments in a pH-dependent manner , Abp140 serves mainly as a crosslinker and visualization tool. While tropomyosins decorate cables uniformly throughout the cell, Abp140 shows compartmentalized binding preferences . The intrinsically disordered nature of juxtanodin contrasts with the domain-specific functionality of Abp140, highlighting the diversity within this protein family .

What is Lifeact and how does it relate to the full-length 71 kDa Abp140 protein?

Lifeact represents the first 17 amino acids of Abp140 and constitutes the minimal F-actin binding domain of the protein . This short peptide maintains sufficient binding affinity for F-actin while eliminating the additional domains present in the full-length protein. When fluorescently tagged, Lifeact effectively labels F-actin structures in living cells across diverse model systems .

Key differences between Lifeact and full-length Abp140 include:

• Binding Pattern: Lifeact (when expressed from the endogenous ABP140 promoter) uniformly decorates actin structures in both mother and bud compartments, unlike the asymmetric decoration observed with full-length Abp140 .

• Signal Quality: Lifeact fusions (particularly with 3xmNeonGreen) provide significantly improved signal-to-background ratios compared to full-length Abp140, enhancing detection of subtle actin structures .

• Functional Domains: Lifeact lacks the tRNA methyltransferase domain present in full-length Abp140, potentially reducing secondary cellular effects when used as an imaging tool .

The methodological approach for implementing Lifeact as an F-actin visualization tool involves:

• Expressing Lifeact-fluorescent protein fusions under control of the endogenous ABP140 promoter

• Optimizing expression levels to prevent disruption of native actin dynamics

• Using appropriate fluorescent tags (3xmNeonGreen shows superior results) to maximize signal intensity

• Applying conventional fluorescence microscopy techniques for live imaging

What experimental advantages does Lifeact offer over full-length Abp140 for studying actin dynamics?

Lifeact provides several significant experimental advantages compared to the full-length 71 kDa Abp140 protein:

• Uniform Decoration: Unlike full-length Abp140, which preferentially decorates mother cell cables, Lifeact uniformly labels actin structures throughout the cell when expressed from the endogenous ABP140 promoter . This eliminates compartment-specific biases in visualization.

• Enhanced Detection Sensitivity: Lifeact-3xmNeonGreen dramatically improves cable detection (approximately 20-fold enhancement) without altering cellular actin organization or dynamics . This improved sensitivity reveals previously undetectable actin structures.

• Visualization of Novel Structures: The superior signal-to-noise ratio enables observation of actin cables growing inward from the cell cortex and dynamically interacting with vacuoles, structures not readily detected using Abp140-GFP .

• Robust Cytokinetic Ring Labeling: Lifeact-3xmNeonGreen effectively decorates the actomyosin ring during cytokinesis, facilitating studies of cell division mechanisms .

• Cross-Organism Applicability: While developed in yeast, Lifeact has been successfully employed in diverse model systems including Arabidopsis thaliana, Caerhabditis elegans, and Schizosaccharomyces pombe , demonstrating its versatility as a research tool.

Experimental data shows that when Lifeact-3xmNeonGreen is expressed at relatively low levels under the control of the native ABP140 promoter, it provides intense labeling of cellular F-actin structures without the detrimental effects observed when Lifeact is overexpressed .

How do intrinsically disordered regions in F-actin binding proteins influence their function and experimental applications?

While Abp140 has not been specifically characterized as intrinsically disordered, the study of juxtanodin (an intrinsically disordered F-actin binding protein) provides valuable insights into this property's impact on protein function . Understanding these principles has important implications for working with various actin-binding proteins:

• Conformational Flexibility: Intrinsically disordered proteins (IDPs) exhibit enhanced structural plasticity, enabling them to adopt multiple conformations and interact with diverse binding partners . This flexibility may facilitate complex formation with different actin structures or regulatory proteins.

• Induced Folding: Upon binding to their targets, IDPs often undergo partial folding. For instance, juxtanodin demonstrates the presence of more compact subpopulations in solution, suggesting conformational changes occur during interactions . This dynamic structuring may enable precise regulation of binding activities.

• Protein Interaction Hubs: The disordered nature of some actin-binding proteins predicts functions as interaction hubs that can link actin filaments to multiple partners . This characteristic enables the formation of complex cytoskeletal networks.

For experimental applications, researchers should consider:

• Using complementary structural characterization techniques (CD spectroscopy, SAXS, and NMR) instead of relying solely on crystallography

• Implementing ensemble optimization analysis to detect subpopulations with different conformational properties

• Interpreting binding studies in the context of potential induced folding upon partner interaction

• Designing experiments to capture potential interactions beyond the primary F-actin binding activity

The intrinsically disordered regions likely contribute to functional versatility, allowing proteins to mediate complex interactions between the actin cytoskeleton and other cellular components .

What is the biological significance of the compartmentalized decoration pattern observed with full-length Abp140?

The asymmetric decoration pattern exhibited by full-length Abp140—preferentially labeling mother cell cables over bud compartment cables—represents an intriguing biological phenomenon with several potential implications:

• Mechanistic Insights: Experiments demonstrate that asymmetric decoration by Abp140 requires F-actin binding , suggesting structural or compositional differences between actin cables in different cellular compartments. These differences may reflect distinct regulatory mechanisms governing cytoskeletal organization during cell division.

• Formin Independence: Studies using integrated Bni1-Bnr1 and Bnr1-Bni1 chimeras demonstrate that the asymmetric cable decoration does not depend on which formin assembles the cables in each compartment . This finding challenges initial hypotheses about the mechanism behind compartmentalized binding.

• Methodological Implications: The compartmentalized binding pattern highlights the importance of choosing appropriate imaging probes. While Abp140-GFP may underrepresent bud compartment cables, mNG-Tpm1 and mNG-Tpm2 robustly decorate cables in both compartments .

• Biological Significance: The differential binding may reflect specialized functions of actin cables in different cellular regions. Cables in the mother cell and bud likely serve distinct purposes during polarized growth and cell division.

What are the optimal conditions for expressing and purifying recombinant 71 kDa F-actin-binding protein for functional studies?

Successful expression and purification of recombinant Abp140 requires careful attention to methodological details. Based on protocols established for similar actin-binding proteins, the following approach is recommended:

Expression Systems and Conditions:

• Bacterial Expression System: E. coli BL21(DE3) or similar strains are suitable for producing the 71 kDa protein, as demonstrated for related actin-binding proteins .

• Vector Design: Include appropriate fusion tags for purification without interfering with actin-binding properties.

• Induction Conditions: IPTG induction at concentrations of 0.1-0.5 mM, at reduced temperatures (16-20°C) to enhance proper folding.

• Expression Duration: Extended expression periods (overnight) at lower temperatures may improve yield of functional protein.

Purification Protocol:

• Initial Capture: Affinity chromatography using appropriate fusion tags.

• Intermediate Purification: Ion exchange chromatography, which has proven effective for actin-binding proteins like cofilin .

• Polishing Step: Size exclusion chromatography to remove aggregates and ensure homogeneity.

Quality Control Metrics:

• Purity Assessment: SDS-PAGE with Coomassie staining should demonstrate ≥95% purity .

• Functional Verification: Co-sedimentation assays with F-actin to confirm binding activity .

• Stability Testing: Thermal shift assays to evaluate protein stability under various buffer conditions.

Storage Recommendations:

• Short-term Storage: At 4°C with antimicrobial agents (100 μg/ml ampicillin and 5 μg/ml chloramphenicol) .

• Long-term Storage: Lyophilization with stabilizers (5% sucrose and 1% dextran) for storage at 4°C , or in solution at -70°C.

• Reconstitution Buffer: 10 mM Tris pH 8.0, 1 mM EGTA, with appropriate stabilizers .

For functional verification, actin co-sedimentation assays provide the most direct demonstration of biological activity, as shown with related actin-binding proteins .

How can researchers optimize fluorescent fusion constructs for visualizing F-actin dynamics?

The design and implementation of fluorescent protein fusions significantly impact visualization quality and experimental outcomes. Based on recent advances, the following optimization strategies are recommended:

Fusion Construct Design:

• Fluorescent Protein Selection: mNeonGreen provides superior brightness for single-molecule detection, while multiple tandem copies (3xmNeonGreen) dramatically enhance signal intensity .

• Fragment Selection: Using Lifeact (first 17 amino acids of Abp140) rather than full-length Abp140 provides more uniform decoration of actin structures throughout the cell .

• Linker Optimization: Incorporating flexible linkers between the actin-binding domain and fluorescent protein reduces steric hindrance.

• Expression Control: Utilizing the endogenous ABP140 promoter prevents overexpression artifacts that can disrupt native actin dynamics .

Comparative Performance Data:
The table below summarizes experimental findings on different fluorescent fusion constructs:

Implementation Protocol:

• Generate integration vectors expressing Lifeact-3xmNG under control of the endogenous ABP140 promoter

• Transform yeast strains using standard protocols

• Verify expression by fluorescence microscopy

• For imaging:

  • Use appropriate exposure settings to minimize photobleaching

  • Consider deconvolution to enhance signal-to-noise ratio

  • Implement time-lapse imaging to capture dynamic changes

This optimized approach has enabled detection of previously unobservable actin structures, including cables growing inward from the cell cortex and dynamically interacting with vacuoles .

What novel insights into actin dynamics have emerged from improved visualization techniques using optimized F-actin binding probes?

The development of enhanced F-actin visualization tools, particularly Lifeact-3xmNeonGreen, has revealed previously undetectable aspects of actin dynamics and organization:

• Inward-Growing Actin Cables: Improved visualization has enabled detection of actin cables that grow inward from the cell cortex toward the cell interior, structures that were not observable using conventional Abp140-GFP . These cables may play roles in intracellular transport and organelle positioning.

• Actin-Vacuole Interactions: Enhanced imaging has revealed dynamic contacts between actin cables and vacuolar membranes . These interactions suggest novel functions for the actin cytoskeleton in regulating vacuole morphology, positioning, and potentially membrane dynamics.

• Cytokinetic Ring Dynamics: Lifeact-3xmNeonGreen robustly decorates the actomyosin ring during cytokinesis , providing improved visualization of this essential structure. This enhanced detection enables more detailed analysis of ring assembly, constriction, and disassembly during cell division.

• Compartmentalized Cytoskeletal Regulation: The discovery that Abp140 preferentially decorates mother cell cables has revealed potential biochemical differences between actin structures in different cellular compartments . These findings suggest specialized regulation of cytoskeletal dynamics during polarized growth.

• Independent Formins Produce Similarly Decorated Cables: Studies using chimeric formins demonstrated that the asymmetric decoration pattern is not dependent on which formin (Bni1 or Bnr1) assembles the cables . This indicates that post-assembly modifications or associated proteins, rather than assembly mechanisms, dictate binding preferences.

These discoveries highlight how technological improvements in visualization tools continue to expand our understanding of fundamental cellular processes. The superior sensitivity of optimized actin probes enables detection of subtle structures and dynamic interactions that were previously unobservable, opening new avenues for research into cytoskeletal organization and function.

How do researchers address technical challenges in studying compartmentalized actin dynamics?

Investigating compartmentalized actin dynamics presents several technical challenges that researchers must overcome:

• Probe-Induced Artifacts: Overexpression of F-actin binding probes can alter native actin dynamics and organization.

  Solution: Express probes from endogenous promoters (like the ABP140 promoter) to maintain appropriate levels . Validate observations using multiple independent approaches.

• Compartmental Bias: Some probes (like Abp140) exhibit preferential binding to specific subcellular regions, potentially misrepresenting actual actin distribution.

  Solution: Implement complementary visualization approaches using different probes (Abp140, tropomyosins, Lifeact) to obtain a comprehensive view . Compare live imaging with fixed-cell phalloidin staining to validate observations.

• Signal-to-Noise Limitations: Weak fluorescent signals from traditional probes may obscure subtle actin structures.

  Solution: Utilize enhanced probes like Lifeact-3xmNeonGreen that provide dramatically improved detection sensitivity . Implement advanced imaging techniques such as deconvolution or structured illumination microscopy.

• Temporal Resolution Constraints: Rapid actin dynamics may be missed due to limitations in imaging speed.

  Solution: Employ high-speed confocal or spinning disk microscopy. Balance exposure time, interval frequency, and total acquisition duration to capture relevant dynamics while minimizing photobleaching.

• Multi-Parameter Analysis: Correlating actin dynamics with other cellular processes requires synchronized multi-channel imaging.

  Solution: Implement dual-color imaging strategies, such as combining Abp140-3xmScarlet with mNG-Tpm1 for ratiometric analysis . Use spectral unmixing when necessary to distinguish overlapping signals.

Experimental approaches that have successfully addressed these challenges include:

• Creating chimeric proteins to investigate domain-specific effects on actin structure and visualization

• Treating cells with CK666 to remove Arp2/3-nucleated patches that can obscure cable staining

• Using latrunculin B as a control to verify F-actin dependent signals

• Implementing ratiometric imaging to quantify relative levels of different actin-binding proteins in distinct cellular compartments

What are the future directions for research utilizing recombinant 71 kDa F-actin-binding protein and its derivatives?

Several promising research directions utilizing Abp140 and its derivatives are emerging:

• Multi-Organism Implementation: While Lifeact has been successfully employed in several model systems, optimized versions like Lifeact-3xmNeonGreen could be adapted for organisms where higher levels of Lifeact expression are detrimental .

• Investigation of Compartmentalized Binding Mechanisms: Further research into the molecular basis of Abp140's preferential decoration of mother cell cables could reveal novel insights into compartmentalized cytoskeletal regulation.

• Analysis of Inward-Growing Cables and Organelle Interactions: The improved visualization enabled by Lifeact-3xmNeonGreen provides opportunities to investigate the functions and regulatory mechanisms of newly detected actin structures, particularly those interacting with organelles .

• Development of Next-Generation Probes: Building on the success of Lifeact-3xmNeonGreen, researchers could develop additional specialized probes that selectively label different subpopulations of actin structures.

• Integration with Advanced Imaging Technologies: Combining optimized actin probes with super-resolution microscopy, light sheet imaging, or correlative light-electron microscopy could further enhance visualization capabilities.