Recombinant Styela plicata Actin, muscle

What distinct muscle actin isoforms have been identified in Styela plicata?

Styela plicata, like other ascidians, possesses at least two distinct muscle actin isoforms: the larval muscle actin and the adult body-wall muscle actin. These isoforms are clearly distinguished by diagnostic amino acids in their sequences. Research has shown that the adult body-wall muscle actin is molecularly distinct from the larval muscle actin, reflecting their different developmental origins and functional roles . Additionally, while not extensively characterized, muscle actin genes expressed in adult heart muscle appear to differ from those expressed in larval tail muscle, suggesting a third possible muscle actin variant .

How does Styela plicata actin expression differ between developmental stages?

The expression of muscle actin genes in Styela follows specific temporal and spatial patterns during development. In Styela clava, a related species, at least four different muscle actin genes (ScTb1, ScTb24, ScTb30, and ScTb12/34) encoding the same actin isoform are expressed in larval tail muscle cells . Some transcripts (ScTb1) are detected at very low levels in eggs and disappear shortly after fertilization, while others show no maternal contribution. Zygotic transcription of these genes first appears during gastrulation, with transcripts gradually accumulating during subsequent neurulation and muscle cell differentiation . These expression patterns reflect the complex regulation of muscle development in ascidians.

What is the significance of α-Smooth Muscle Actin (α-SMA) expression in Styela plicata tissues?

Recent immunofluorescence studies have revealed α-SMA expression in the cells lining the vessels of the tunic in Styela plicata . This is particularly significant as it demonstrates conservation of smooth muscle actin expression in specific tissues. The tunic, situated outside the epidermis, contains blood vascular lacunae with a fibrous structure. The identification of α-SMA in these cells suggests functional contractile mechanisms in the circulatory system of ascidians, providing insights into the evolution of vascular smooth muscle in chordates .

What purification challenges are specific to recombinant ascidian muscle actin?

Purifying recombinant muscle actin from Styela plicata presents several challenges. Actin's tendency to polymerize during purification often complicates isolation procedures. Effective strategies include using ATP and calcium chelators (EGTA) during initial extraction to maintain actin in its monomeric state. Affinity tags (such as His6 or GST) can facilitate purification, though they might affect protein function and should be removable by protease cleavage. DNase I affinity chromatography, leveraging actin's natural binding to DNase I, provides a tag-free approach for obtaining highly pure G-actin. Multiple stages of purification are typically required, including ion exchange chromatography followed by size exclusion chromatography to separate monomeric from oligomeric actin forms.

How can researchers confirm the proper folding and functionality of recombinant Styela plicata muscle actin?

Verifying proper folding and functionality of recombinant Styela plicata muscle actin requires multiple complementary approaches:

• Polymerization assays: Monitoring the kinetics of actin polymerization in the presence of ATP and divalent cations using pyrene-labeled actin or light scattering

• DNase I binding assays: Properly folded G-actin inhibits DNase I activity

• ATPase activity measurements: Functional actin exhibits specific ATPase activity during polymerization

• Electron microscopy or fluorescence microscopy: Direct visualization of actin filament formation using labeled actin

• Circular dichroism spectroscopy: Assessment of secondary structure to confirm proper protein folding

Additionally, binding assays with known actin-binding proteins from related species can confirm the functional integrity of the recombinant protein.

How do the molecular characteristics of Styela plicata muscle actin compare with other chordate actins?

Molecular phylogenetic analysis of actin sequences suggests a close relationship between ascidian and vertebrate actins. The comparative analysis indicates that the chordate ancestor evolved "chordate-type" cytoplasmic and muscle actins before diverging into vertebrates and urochordates . When compared to vertebrate muscle actins, Styela plicata muscle actin shows high conservation in functional domains involved in ATP binding, divalent cation coordination, and polymerization interfaces.

Table 1: Comparative Analysis of Key Molecular Features in Chordate Actins

What evolutionary insights can be gained from studying Styela plicata muscle actin genes?

The study of Styela plicata muscle actin genes provides valuable insights into chordate evolution. Phylogenetic analysis suggests that vertebrate muscle actin isoforms evolved after the separation of vertebrates and urochordates, while the basic distinction between cytoplasmic and muscle actins predates this divergence . Research on muscle actin genes has also been used to investigate the mechanism of muscle cell regression during the evolution of anural development, suggesting that regression of muscle cell differentiation is mediated by changes in the structure of muscle actin genes rather than in the trans-acting regulatory factors required for their expression . This evolutionary perspective helps understand how tissue-specific actins diversified during chordate evolution.

How does the gene organization of muscle actin in Styela plicata contribute to its expression patterns?

The genomic organization of muscle actin genes in Styela plicata appears to play a crucial role in their expression patterns. In related ascidian species like Halocynthia roretzi, muscle actin genes are organized in gene clusters, including tandem clusters and bidirectional promoters . This organization likely facilitates coordinated expression and enables the rapid synthesis of large amounts of actin during muscle cell differentiation. The presence of multiple muscle actin genes encoding similar or identical proteins is a common feature in ascidians, suggesting that gene duplication and diversification of regulatory elements have been important in the evolution of muscle-specific gene expression in these organisms .

How can recombinant Styela plicata muscle actin be used to study innate immune functions in ascidians?

Recent research has revealed an unexpected connection between muscle actin expression and innate immunity in ascidians. Studies have shown that Toll-like receptors (TLRs), key components of the innate immune system, are expressed in various tissues of ascidians, including those expressing α-Smooth Muscle Actin . Recombinant Styela plicata muscle actin can be used as a tool to investigate potential interactions between the cytoskeleton and immune response elements, particularly in the tunic vasculature where both α-SMA and TLR2 have been detected .

Experimental approaches might include:

• Co-immunoprecipitation studies using recombinant actin to identify binding partners involved in immune signaling

• In vitro reconstitution of actin-based structures in the presence of immune stimulants

• Localization studies using fluorescently labeled recombinant actin during immune challenges

• Actin polymerization assays in the presence of hemocyte extracts to detect regulatory molecules

These applications would provide insights into the evolutionary conservation of cytoskeletal involvement in immune functions across chordates.

What techniques can be used to study the interaction between recombinant Styela plicata muscle actin and other contractile proteins?

Investigating interactions between recombinant Styela plicata muscle actin and other contractile proteins requires a multi-faceted approach:

• Co-sedimentation assays: Mixing recombinant actin with myosin or tropomyosin and analyzing the pellet and supernatant fractions after high-speed centrifugation.

• Surface plasmon resonance (SPR): Determining binding kinetics and affinities between immobilized actin and flowing contractile proteins.

• Microscale thermophoresis (MST): Measuring interactions in solution by detecting changes in thermophoretic mobility upon binding.

• In vitro motility assays: Observing movement of fluorescently labeled actin filaments over a surface coated with myosin motor proteins.

• FRET (Förster Resonance Energy Transfer): Using fluorescently labeled actin and binding partners to detect proximity-dependent energy transfer.

• Cryo-electron microscopy: Visualizing the structural details of actin-myosin or actin-tropomyosin complexes at near-atomic resolution.

These techniques allow researchers to determine both the biochemical and structural aspects of interactions between ascidian muscle actin and its binding partners.

How can Styela plicata muscle actin be used to study mechanisms of muscle development in ascidian adults?

Adult body-wall muscle actin genes from ascidians like Styela plicata represent valuable tools for investigating muscle development in adult ascidians . These genes can be used to:

• Create reporter constructs with the regulatory regions of adult muscle actin genes to track muscle cell differentiation in vivo.

• Develop tissue-specific expression systems for genetic manipulation of adult muscle cells.

• Analyze the transcriptional regulatory networks controlling adult muscle development through chromatin immunoprecipitation (ChIP) and promoter analysis studies.

• Compare expression patterns of larval vs. adult muscle actin genes during metamorphosis to understand the transition between these developmental programs.

• Study the role of epigenetic modifications in the switch from larval to adult muscle gene expression.

This research contributes to our understanding of how distinct muscle tissues develop in chordates and how developmental programs are remodeled during metamorphosis.

What strategies can overcome protein aggregation issues during recombinant Styela plicata muscle actin production?

Protein aggregation is a common challenge when producing recombinant actin. Several strategies can mitigate this issue:

• Co-expression with molecular chaperones: Including folding assistants like GroEL/ES or DnaK/J/GrpE in bacterial systems, or appropriate chaperones in eukaryotic systems.

• Optimization of induction conditions: Lower temperatures (15-20°C), reduced inducer concentrations, and slower induction rates often improve proper folding.

• Fusion tags that enhance solubility: MBP (maltose-binding protein) or SUMO tags can significantly enhance solubility while allowing subsequent tag removal.

• Addition of stabilizing agents: Including ATP, divalent cations (Mg²⁺), and specific osmolytes (trehalose, glycerol) in lysis and purification buffers.

• Arginine suppression method: Adding L-arginine to purification buffers (typically 0.5-1M) to suppress aggregation of partially folded intermediates.

• On-column refolding protocols: Immobilizing the denatured protein on affinity columns before gradually removing denaturants.

These approaches can be combined and optimized based on specific expression systems and the properties of the particular Styela plicata actin isoform being produced.

How can researchers distinguish between different actin isoforms when studying Styela plicata?

Distinguishing between different actin isoforms in Styela plicata requires a combination of techniques:

• Isoform-specific antibodies: Generating antibodies against unique epitopes, particularly in the N-terminal region where most isoform-specific differences occur.

• Mass spectrometry: Peptide mass fingerprinting and tandem MS can identify diagnostic peptides that differ between isoforms.

• 2D gel electrophoresis: Separation based on both isoelectric point and molecular weight can resolve very similar actin isoforms.

• RT-PCR with isoform-specific primers: Targeting unique nucleotide sequences in the coding or untranslated regions.

• RNA-seq analysis: Mapping reads to specific isoform transcripts for quantitative assessment of expression levels.

• In situ hybridization: Using isoform-specific probes to determine tissue-specific expression patterns.

These approaches provide complementary information about the identity, abundance, and localization of different actin isoforms during development and in adult tissues.

What experimental design considerations are important when studying the effects of environmental stressors on Styela plicata muscle actin expression?

When designing experiments to study how environmental stressors affect muscle actin expression in Styela plicata, researchers should consider:

• Acclimation periods: Allow sufficient time for specimens to acclimate to laboratory conditions before applying stressors (typically 1-2 weeks).

• Appropriate controls: Include proper controls for handling stress and time-matched sampling to account for natural temporal variations in gene expression.

• Stressor intensity and duration: Apply stressors at ecologically relevant levels and durations, based on natural conditions. For example, in temperature stress studies, consider the range observed in the relevant data from Table 1 in search result :

Table 2: Relevant Environmental Parameters for Styela plicata Experimental Design

• Multiple tissue sampling: Sample different muscle tissues (larval tail muscle, adult body-wall muscle, heart muscle) as they may respond differently.

• Time-course sampling: Collect samples at multiple time points to capture immediate, intermediate, and long-term transcriptional responses.

• Integrated omics approach: Combine transcriptomics with proteomics to distinguish between transcriptional and post-transcriptional regulation of actin expression.

• Functional validation: Include assays of muscle contractility or actin polymerization to correlate gene expression changes with functional outcomes.

These considerations ensure that experiments generate biologically meaningful data about stress responses in Styela plicata muscle tissues.

How might CRISPR/Cas9 genome editing be applied to study Styela plicata muscle actin function?

CRISPR/Cas9 genome editing offers powerful approaches to study muscle actin function in Styela plicata:

• Targeted gene knockouts: Creating null mutations in specific actin isoforms to determine their precise functions in development and physiology.

• Regulatory element analysis: Editing promoter or enhancer sequences to identify critical regions for tissue-specific or stage-specific expression.

• Fluorescent tagging: Inserting fluorescent protein sequences to track the expression and localization of actin isoforms in living tissues.

• Domain swapping: Replacing specific domains or motifs between different actin isoforms to determine their functional significance.

• Humanized variants: Creating chimeric genes with vertebrate actin sequences to study evolutionary conservation of function.

These approaches require optimization of microinjection techniques for Styela plicata embryos and efficient delivery methods for CRISPR components, but they promise unprecedented insights into actin function in this important model organism.

What are the potential applications of recombinant Styela plicata muscle actin in comparative biomechanics research?

Recombinant Styela plicata muscle actin provides unique opportunities for comparative biomechanics research:

• Evolutionary biomechanics: Comparing the mechanical properties of filaments formed by actins from different taxonomic groups to understand how molecular changes affect force generation.

• Structure-function relationships: Analyzing how specific amino acid differences between ascidian and vertebrate actins affect interactions with myosin and force production.

• Hybrid systems: Creating in vitro motility assays with mixtures of proteins from different species to identify compatibility and constraints in molecular co-evolution.

• Temperature adaptation studies: Examining how actins from species adapted to different thermal environments perform across temperature ranges.

• Biomimetic applications: Utilizing unique properties of ascidian actins in engineered nanomaterials or biosensors.

These applications contribute to our understanding of how molecular evolution shapes the mechanical properties of contractile systems across the animal kingdom.

How can systems biology approaches integrate Styela plicata muscle actin research into broader understanding of chordate muscle evolution?

Systems biology approaches can contextualize Styela plicata muscle actin research within the broader framework of chordate muscle evolution:

• Comparative genomics and transcriptomics: Analyzing the complete repertoire of contractile and regulatory proteins across chordate lineages to identify conserved and divergent elements.

• Protein interaction networks: Mapping the interactome of muscle actins across species to identify evolutionary changes in protein-protein interactions.

• Mathematical modeling: Developing models that integrate biochemical, structural, and mechanical data to predict how molecular changes affect muscle function.

• Evolutionary developmental biology: Integrating gene expression data with morphological development to understand how changes in actin genes relate to innovations in muscle structure.

• Phylogenetic inference: Using actin sequence and functional data to refine our understanding of chordate phylogeny and the timing of evolutionary innovations.

These integrative approaches will help place the specific findings from Styela plicata research into an evolutionary context, illuminating the molecular basis for the diversity of muscle forms and functions across chordates.