S100A6 Mouse

What is the molecular structure of mouse S100A6 and how does it compare to human S100A6?

Mouse S100A6 is an 89 amino acid protein containing two calcium-binding EF-hand domains located at amino acids 12-47 and 48-83. As a member of the S100 family, it functions both as a calcium sensor and a calcium signal modulator in cells. Mouse S100A6's structure is highly conserved across species, with 96% amino acid identity to human S100A6 and 99% identity to rat S100A6 .

The protein exhibits calcium-dependent conformational changes that enable interactions with various target proteins. In its dimeric form, each S100A6 monomer can bind two calcium ions, allowing the protein to respond sensitively to changes in intracellular calcium concentrations.

Which tissues and cell types express S100A6 in mice, and how can this expression be reliably detected?

S100A6 is expressed in multiple mouse tissues and cell types including:

• Hematopoietic stem cells (particularly abundant in long-term HSCs)

• Neurons

• Endothelial cells

• Fibroblasts (including NIH-3T3 cell line)

• Glandular epithelia

• Monocytes/macrophages (including RAW 264.7 cell line)

• Kidney epithelial cells in convoluted tubules

Detection methods include:

• Western blot: Using specific antibodies such as Sheep Anti-Human/Mouse S100A6 Antibody, which detects a band at approximately 10 kDa under reducing conditions

• Immunohistochemistry: Paraffin-embedded sections can be subjected to heat-induced epitope retrieval and stained with S100A6 antibodies

• Simple Western™: An automated capillary-based detection method showing bands at approximately 7 kDa

• RT-qPCR: For analyzing S100A6 transcript levels across different cell populations

For optimal detection specificity, validation with S100A6 knockout controls is essential, as demonstrated by the absence of S100A6 bands in S100A6 knockout HEK293T cell lines .

How does S100A6 regulate hematopoietic stem cell function and what signaling pathways are involved?

S100A6 functions as a critical regulator of hematopoietic stem cell (HSC) self-renewal and survival through several mechanisms:

• Akt Pathway Regulation: S100A6 governs the Akt activation pathway, which is essential for HSC survival. The relationship between S100A6 and Akt is demonstrated by the ability of Akt activator SC79 to rescue colony formation defects in S100A6-deficient bone marrow cells .

• Antiapoptotic Effects: S100A6 provides significant antiapoptotic protection in murine HSCs. S100A6KO mice show increased Annexin V+/DAPI+ expression in long-term HSCs, indicating elevated apoptosis .

• Mitochondrial Function: S100A6 regulates:

  • Mitochondrial calcium levels

  • Respiratory metabolism, particularly aerobic respiration

  • Expression of mitochondrial proteins like isocitrate dehydrogenase (IDH2)

• Protein Quality Control: S100A6 influences the HSP90 protein pathway, which is critical for proper protein folding and elimination of toxic protein aggregates .

These mechanisms collectively contribute to HSC maintenance and regenerative capacity, as evidenced by transplantation studies showing severely impaired reconstitution ability of S100A6-deficient HSCs .

What phenotypes are observed in S100A6 knockout mouse hematopoietic systems?

S100A6 conditional knockout mice (S100A6KO) in the hematopoietic system display several significant phenotypes:

What are the most effective methods for isolating and studying S100A6 in mouse hematopoietic stem cells?

For isolating and studying mouse hematopoietic stem cells (HSCs) to investigate S100A6 function:

A. HSC Isolation Protocol:

• Bone marrow extraction from femurs and tibias

• Lineage depletion using magnetic beads

• Fluorescence-activated cell sorting (FACS) with increasing specificity:

  • Base population: LSK (Lineage−Sca-1+c-Kit+)

  • HSC enrichment: LSKCD150+CD48−

  • Purified LT-HSCs: LSKCD150+CD48−CD34−Flt3−

  • Most stringent purification: LSKCD150+CD48−CD34−Flt3−CD9HiESAM+

B. Functional Assays:

• Transplantation assays:

  • Competitive transplantation with 50 purified LT-HSCs

  • Assessment of donor-derived cells in peripheral blood and bone marrow compartments

  • Serial transplantation to test self-renewal capacity

• Colony formation assays:

  • Methylcellulose cultures with defined cytokine combinations

  • Analysis of colony numbers and types

  • Testing rescue with Akt pathway activators like SC79

• Molecular analyses:

  • RNA-sequencing of purified HSC populations

  • Proteomics analysis to identify differentially expressed proteins

  • Network analysis to reveal affected pathways

• Apoptosis assessment:

  • Annexin V and DAPI staining of freshly isolated HSCs

  • Flow cytometric quantification of apoptotic cell percentages

How can researchers generate and validate S100A6 knockout mouse models for hematopoietic research?

Generation of S100A6 Conditional Knockout Mice:

• Gene targeting strategy:

  • Create a floxed S100A6 allele with loxP sites flanking critical exons

  • Cross with mice harboring tissue-specific Cre recombinase (e.g., Vav-Cre for hematopoietic-specific deletion)

  • Breed to homozygosity to obtain complete knockout in target tissues

• Breeding scheme:

  • Cross flox/flox S100A6 mice with Vav-Cre transgenic mice

  • Identify Vav-Cre;S100A6 mice (mutant) and Vav-negative littermates (control)

  • Further breeding of ΔΔ Vav-Cre;S100A6 with flox/flox Vav-Cre;S100A6 for multiple generations

Validation Methods:

• Genotyping:

  • PCR confirmation of floxed alleles and Cre transgene presence

  • Verification of deletion through PCR of excised regions

• Expression analysis:

  • RNA-seq or qRT-PCR to confirm reduced S100A6 transcript levels

  • Western blot to verify absence of S100A6 protein

  • In the RNA-seq data, S100A6 should rank among the most downregulated genes (p<0.05)

• Functional validation:

  • Flow cytometric analysis of HSC compartments

  • Transplantation assays to assess HSC function

  • Colony formation assays to test progenitor activity

• Controls:

  • Use age-matched (8-16 weeks) littermate controls

  • Include both males and females in experiments

  • Randomize experimental groups

How can researchers distinguish between intracellular and extracellular functions of S100A6 in mouse models?

S100A6 functions both intracellularly and extracellularly through distinct mechanisms that can be experimentally separated:

Intracellular Function Assessment:

• Subcellular fractionation:

  • Separate cytosolic, mitochondrial, nuclear, and membrane fractions

  • Analyze S100A6 distribution by Western blot

• Intracellular interaction analysis:

  • Co-immunoprecipitation to identify binding partners (e.g., S100B, SGT1)

  • Proximity ligation assays to visualize protein interactions in situ

• Calcium-dependent binding:

  • In vitro binding assays with varying calcium concentrations

  • Use of calcium chelators (BAPTA-AM) to disrupt calcium-dependent interactions

Extracellular Function Assessment:

• Secretion analysis:

  • Measure S100A6 in conditioned media from cultured cells

  • Study the noncanonical secretion pathway using specific inhibitors

• RAGE-mediated signaling:

  • Add recombinant S100A6 protein to cultures

  • Block RAGE using neutralizing antibodies or soluble RAGE

  • Measure downstream effects on apoptosis and signal transduction

• Transplantation strategies:

  • Compare transplantation of S100A6KO cells into wild-type recipients (testing cell-intrinsic effects)

  • Compare wild-type cells into S100A6KO recipients (testing microenvironment effects)

  • Analyze effects of S100A6-containing conditioned media on HSC function

What approaches can be used to study the relationship between S100A6 and mitochondrial function in mouse hematopoietic stem cells?

The relationship between S100A6 and mitochondrial function in HSCs can be investigated through:

• Mitochondrial respiration analysis:

  • Measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)

  • Assess basal respiration, ATP production, maximal respiratory capacity, and spare capacity

  • Compare S100A6-deficient versus wild-type HSCs

• Mitochondrial membrane potential:

  • Use fluorescent dyes like TMRM or JC-1

  • Perform flow cytometry or live-cell imaging

  • Correlate with S100A6 expression levels

• Calcium dynamics:

  • Employ mitochondria-targeted calcium indicators

  • Analyze calcium flux between cytosol and mitochondria

  • Determine how S100A6 mediates these processes

• Proteomic analysis:

  • Focus on mitochondrial fraction proteins

  • Identify differentially expressed proteins between WT and S100A6KO HSCs

  • Pay particular attention to IDH2, which appears downregulated in S100A6KO

• Reactive oxygen species (ROS) assessment:

  • Measure mitochondrial and cytosolic ROS levels

  • Determine if S100A6 influences ROS production or detoxification

  • Connect ROS levels to HSC functional outcomes

• Mitochondrial dynamics:

  • Analyze mitochondrial morphology (fission/fusion balance)

  • Assess mitophagy rates and mitochondrial turnover

  • Quantify mitochondrial mass and distribution

What are common technical challenges in S100A6 detection, and how can they be overcome?

Challenge 1: Variable molecular weight detection

• S100A6 can appear at different molecular weights (7-10 kDa) depending on gel systems

• Solution: Include appropriate positive controls (e.g., NIH-3T3 or RAW 264.7 lysates) and validate the specific band pattern with knockout controls

Challenge 2: Cross-reactivity with other S100 family members

• S100 family proteins share structural similarities

• Solution: Validate antibody specificity using:

  • S100A6 knockout samples as negative controls

  • Western blots comparing parental and S100A6 knockout HEK293T cell lines

  • GAPDH as loading control

Challenge 3: Tissue-specific expression variations

• Expression levels vary significantly between tissues and conditions

• Solution:

  • Optimize antibody concentration for each tissue type

  • For immunohistochemistry, use heat-induced epitope retrieval

  • For Western blot, adjust protein loading (e.g., 0.2 mg/mL for NIH-3T3 and RAW 264.7)

Challenge 4: Detection method limitations

• Different applications require specific protocols

• Solution:

  • For Western blot: Use reducing conditions and Immunoblot Buffer Group 1 or 8

  • For IHC: Apply 3 μg/mL antibody overnight at 4°C after proper antigen retrieval

  • For Simple Western: Use 10 μg/mL antibody with 12-230 kDa separation system

How can researchers assess whether functional effects attributed to S100A6 are specific rather than due to compensatory mechanisms?

To ensure observed effects are specifically due to S100A6 rather than compensatory mechanisms:

• Comprehensive S100 family analysis:

  • Assess expression levels of other S100 family members in S100A6KO models

  • Determine if any family members show compensatory upregulation

  • RNA-seq data can identify potential compensatory mechanisms

• Rescue experiments:

  • Re-introduce wild-type S100A6 into knockout cells

  • Use inducible expression systems to control timing and levels

  • Compare functional rescue with mutant versions (e.g., calcium-binding mutants)

• Pathway-specific validation:

  • If Akt pathway is implicated, use specific Akt inhibitors or activators

  • Confirm that Akt activator SC79 rescues phenotypes in S100A6KO cells

  • Test whether inhibition of compensatory pathways restores the knockout phenotype

• Temporal control of knockout:

  • Use inducible Cre systems to delete S100A6 at different developmental timepoints

  • Distinguish between developmental versus homeostatic requirements

  • Assess acute versus chronic effects of S100A6 loss

• Combined knockouts:

  • Generate double knockouts of S100A6 with potential compensatory proteins

  • Assess whether phenotypes are exacerbated in double knockouts

  • Target specific pathway components to confirm mechanistic relationships