Recombinant Human Actin-binding LIM protein 2 (ABLIM2)

What is Actin-binding LIM protein 2 (ABLIM2) and what are its key functional domains?

ABLIM2 is a member of the actin-binding LIM protein family that plays crucial roles in cytoskeletal organization. It contains multiple LIM domains that are critical for its function. LIM domains are zinc-finger protein interaction domains composed of approximately 55 amino acids with a characteristic arrangement of cysteine and histidine residues that coordinate zinc ions.

The protein structure includes:

• Multiple LIM domains responsible for protein-protein interactions

• An actin-binding domain that enables direct interaction with the cytoskeleton

• Zinc-binding regions that maintain structural integrity

Similar to other LIM domain proteins such as hhLIM, the second LIM domain appears to play a particularly important role in actin bundling and cytoskeletal stabilization . ABLIM2's ability to bind both actin filaments and zinc ions enables it to serve as a scaffold that connects the actin cytoskeleton to various signaling pathways .

What cellular functions and pathways involve ABLIM2?

ABLIM2 participates in several key cellular pathways and functions:

Evidence from studies on related LIM proteins suggests that ABLIM2 likely increases actin cytoskeleton stability by promoting bundling of actin filaments . The zinc ion binding property also suggests a role in transcriptional regulation or protein complex formation .

How is ABLIM2 expression regulated in different tissues?

The regulation of ABLIM2 expression involves both genetic and epigenetic mechanisms. Recent studies have identified that DNA methylation plays a significant role in regulating ABLIM2 expression. Specifically, an intragenic CpG site in ABLIM2 shows differential methylation patterns correlated with expression levels. In cardiac tissue, this CpG was significantly hypermethylated at elevated temperatures (38.9°C) and negatively correlated with heart weight .

Regulatory mechanisms may include:

• Transcriptional regulation through tissue-specific transcription factors

• Epigenetic regulation via DNA methylation patterns

• Post-transcriptional regulation through micro-RNAs

• Post-translational modifications affecting protein stability

While comprehensive tissue-specific expression data for human ABLIM2 is still emerging, understanding these patterns is crucial for researchers designing tissue-specific experiments.

What are the current methodologies for studying ABLIM2's role in actin cytoskeleton remodeling?

To investigate ABLIM2's role in actin cytoskeleton remodeling, researchers typically employ several complementary approaches:

• Fluorescence microscopy with co-localization studies:

  • Express GFP-tagged ABLIM2 in cell lines (such as C2C12)

  • Co-stain for F-actin using fluorescent phalloidin

  • Analyze co-localization patterns to determine association with actin structures

• Actin stability assays:

  • Overexpress ABLIM2 in cell models

  • Challenge with actin depolymerizing agents (e.g., cytochalasin B)

  • Measure the rate of actin depolymerization compared to controls

• In vitro binding and bundling assays:

  • Use purified recombinant ABLIM2 and F-actin

  • Perform low-speed co-sedimentation assays to assess bundling activity

  • Directly observe F-actin bundles formed in the presence of ABLIM2

Based on studies of similar LIM proteins, ABLIM2 likely increases actin cytoskeleton stability by promoting the bundling of actin filaments, similar to what has been observed with hhLIM protein .

How can researchers effectively study the effects of ABLIM2 mutations on protein function?

To study the effects of ABLIM2 mutations on protein function, researchers should consider a systematic approach:

• Domain-specific mutational analysis:

  • Create truncated mutants focusing on individual LIM domains

  • Replace key cysteine residues with serine in zinc-finger motifs

  • Express these mutants in appropriate cell lines and assess their effects

• Functional assays for mutant proteins:

  • Actin binding assays comparing wild-type and mutant proteins

  • Actin bundling assays using low-speed co-sedimentation

  • Cell-based assays examining effects on cytoskeletal organization

• Structural analysis:

  • Determine how mutations affect protein folding and stability

  • Assess impacts on zinc coordination in LIM domains

  • Examine effects on protein-protein interactions

When analyzing LIM domain functionality, focus particularly on the second LIM domain, as studies of related proteins suggest this domain is critical for F-actin bundling activity .

What is the role of ABLIM2 in developmental processes and associated disorders?

ABLIM2 has emerging implications in developmental processes and certain disorders:

• Neurodevelopment:

  • ABLIM2's involvement in axon guidance pathways suggests a role in neural development

  • The protein may regulate neuronal migration and axon pathfinding

• Developmental disorders:

  • Exome sequencing studies have identified ABLIM2 variants in infants with sacral agenesis

  • Compound heterozygous events in ABLIM2 have been reported in developmental disorders

• Cardiac development:

  • DNA methylation studies indicate potential roles in cardiac tissue development

  • An intragenic CpG in ABLIM2 shows hypermethylation patterns that negatively correlate with heart weight

The research on ABLIM2's developmental roles is still evolving, and further investigation is needed to fully characterize its contributions to these processes and associated disorders.

What are the optimal conditions for expressing and purifying recombinant human ABLIM2?

For optimal expression and purification of recombinant human ABLIM2:

• Expression systems:

  • Bacterial systems (E. coli): Suitable for producing the protein for in vitro assays

  • Mammalian systems (HEK293, CHO cells): Preferred for studies requiring post-translational modifications

  • Insect cell systems (Sf9, Hi5): Useful for higher yields of properly folded protein

• Expression optimization:

  • Codon optimization for the chosen expression system

  • Temperature optimization (typically lower temperatures of 16-25°C for E. coli)

  • Induction conditions adjusted to minimize inclusion body formation

• Purification strategy:

  • Affinity chromatography using His-tag or GST-tag

  • Ion exchange chromatography for further purification

  • Size exclusion chromatography as a final polishing step

• Buffer considerations:

  • Use buffers containing zinc (10-50 μM ZnCl₂) to maintain LIM domain integrity

  • Include reducing agents (DTT or β-mercaptoethanol) to prevent oxidation of cysteine residues

  • Stabilizing agents such as glycerol (10-20%) may improve protein stability

Storage recommendations include aliquoting the purified protein and storing at -80°C with 50% glycerol to prevent freeze-thaw cycles that can reduce activity .

What techniques are most effective for detecting and quantifying ABLIM2 in experimental samples?

For effective detection and quantification of ABLIM2:

When using antibodies, polyclonal antibodies generated against recombinant human ABLIM2 protein (amino acids 222-521) have shown good results for multiple applications. For optimal results in immunohistochemistry, dilutions in the range of 1:20 - 1:200 are recommended, though optimal conditions should be determined empirically for each experimental system .

How can researchers effectively use RNA interference to study ABLIM2 function?

For effective RNA interference studies of ABLIM2:

• Selection of RNAi approach:

  • siRNA: For transient knockdown experiments

  • shRNA: For stable knockdown via lentiviral vectors

  • CRISPR-Cas9: For complete gene knockout studies

• Design considerations for siRNA/shRNA:

  • Target multiple sites within the ABLIM2 transcript

  • Use a set of 4 different siRNA constructs to increase chances of effective knockdown

  • Include appropriate negative controls (scrambled sequences)

• Delivery methods:

  • Lipid-based transfection for cell lines with good transfection efficiency

  • Lentiviral vectors for difficult-to-transfect cells or for stable expression

  • Electroporation as an alternative method for certain cell types

• Validation of knockdown:

  • qRT-PCR to confirm reduction in mRNA levels

  • Western blotting to verify protein depletion

  • Functional assays to assess phenotypic changes

When using lentiviral-based systems, commercially available ABLIM2 siRNA/shRNA sets typically include 4 constructs targeting different regions of the transcript. At least one of these constructs should give >70% knockdown efficiency in cells with >80% transfection efficiency. For optimal results, use transfection concentrations ≥5 nM and assess knockdown 48 hours post-transfection .

How does ABLIM2 function compare to other actin-binding LIM proteins?

ABLIM2 shares functional similarities with other actin-binding LIM proteins, but with distinct characteristics:

• Structural comparisons:

  • Like hhLIM, ABLIM2 contains multiple LIM domains

  • ABLIM2's second LIM domain likely plays a critical role in actin bundling

  • The specific arrangement of LIM domains differs between family members

• Functional similarities and differences:

  • Similar to hhLIM, ABLIM2 likely stabilizes actin filaments and delays depolymerization

  • ABLIM2 appears to function as an actin-bundling protein

  • Different family members may show tissue-specific expression patterns

• Evolutionary conservation:

  • LIM domains are highly conserved structural motifs

  • Actin-binding mechanisms show similarities across family members

  • The number and arrangement of domains vary between proteins

Studies on hhLIM provide a useful model for understanding ABLIM2 function. Research has shown that hhLIM accumulates in the cytoplasm and colocalizes with F-actin, significantly stabilizing actin filaments and delaying depolymerization induced by cytochalasin B treatment. Expression of hhLIM induced significant changes in actin cytoskeleton organization, resulting in fewer but thicker actin bundles .

What are the emerging roles of ABLIM2 in disease processes?

ABLIM2 has several emerging roles in disease processes that represent important research directions:

• Developmental disorders:

  • Exome sequencing has identified ABLIM2 variants in infants with sacral agenesis

  • Compound heterozygous events in ABLIM2 may contribute to developmental abnormalities

• Cardiac pathology:

  • DNA methylation studies suggest ABLIM2 may play a role in cardiac development

  • Hypermethylation of an intragenic CpG in ABLIM2 negatively correlates with heart weight

• Neurological disorders:

  • Given ABLIM2's role in axon guidance pathways, it may contribute to neurological conditions

  • Dysregulation of cytoskeletal proteins including ABLIM2 could impact neuronal migration and function

• Cancer progression:

  • As an actin-binding protein, ABLIM2 may influence cancer cell motility and metastasis

  • Altered expression could potentially impact tumor cell invasion capabilities

These emerging roles highlight the importance of continued research on ABLIM2 in various disease contexts, with potential implications for diagnostic and therapeutic approaches.

What experimental models are most suitable for studying ABLIM2 function in vivo?

For in vivo studies of ABLIM2 function, several experimental models offer distinct advantages:

• Mouse models:

  • Genetic knockout or knockdown models using CRISPR-Cas9 or shRNA technology

  • Conditional knockout models for tissue-specific analysis

  • Can be used with commercially available Ablim2-set siRNA/shRNA lentivectors

• Zebrafish models:

  • Rapid development and optical transparency

  • Amenable to gene editing and high-throughput screening

  • Particularly useful for studying neuronal and cardiac development

• Cell culture models:

  • C2C12 myoblast cells are useful for studying ABLIM2's role in the actin cytoskeleton

  • Neuronal cell lines for investigating axon guidance functions

  • Primary cells from specific tissues for context-dependent studies

• Drosophila models:

  • Genetic tractability and rapid generation time

  • Useful for studying conserved developmental pathways

  • Powerful for genetic interaction studies

When selecting an experimental model, consideration should be given to the specific research question, available genetic tools, and relevance to human biology. For studies focused on protein-protein interactions and cytoskeletal functions, cell culture systems using fluorescently tagged constructs offer advantages in visualization and manipulation. For developmental studies, vertebrate models like mice or zebrafish provide more relevant physiological contexts.