TSPAN7 Human

What is the basic structure of human TSPAN7 and how does it compare to other tetraspanins?

TSPAN7, like other tetraspanins, has four transmembrane domains, a small extracellular loop (SEL), a large extracellular loop (LEL), and intracellular N- and C-terminal tails. The protein contains several key functional regions that distinguish it from other tetraspanins, including interaction sites at E115 in the LEL, amino acids 190-199 (TVAATKVNQK) also in the LEL, and amino acids 53-57 (ENSTN) in the SEL . These regions are fully conserved between mouse and human TSPAN7, highlighting their evolutionary importance. While E115 is conserved in the related protein TSPAN6, the other two regions (aa53-57 and aa190-199) are not conserved in either TSPAN6 or TSPAN4, suggesting unique functional properties of TSPAN7 .

What are the known protein interaction partners of TSPAN7 and their functional significance?

TSPAN7 directly interacts with several key proteins that mediate its cellular functions:

• PICK1 (Protein Interacting with C Kinase 1): TSPAN7 binds to the PDZ domain of PICK1, which is important for regulating AMPA receptor trafficking .

• GluA2/3 (AMPA receptor subunits): TSPAN7 associates with these glutamate receptor subunits and regulates their trafficking to synapses .

• β1-integrin: This association may mediate TSPAN7's effects on actin filament organization and cytoskeletal dynamics .

These interactions collectively contribute to TSPAN7's role in dendritic spine formation, synaptic stability, and normal synaptic transmission in neurons .

How does TSPAN7 contribute to neuronal development and synaptic function?

TSPAN7 plays crucial roles in neuronal development and synaptic function through multiple mechanisms:

• Dendritic spine formation: TSPAN7 promotes filopodia and dendritic spine formation in cultured hippocampal neurons .

• Synaptic stability: It is required for maintaining stable dendritic spines and normal synaptic transmission .

• AMPA receptor trafficking: TSPAN7 regulates the association between PICK1 and GluA2/3, thereby controlling AMPA receptor trafficking to synapses. This mechanism is essential for synaptic plasticity and function .

• Cytoskeletal regulation: TSPAN7 may affect actin filament organization by binding to PI4K and/or through its interaction with β1-integrin .

Methodologically, these functions have been studied using cultured hippocampal neurons with both overexpression and knockdown approaches, combined with electrophysiological recordings to assess synaptic transmission .

What mutations in TSPAN7/TM4SF2 are associated with intellectual disability, and what are their functional consequences?

Several mutations in the TM4SF2 gene encoding TSPAN7 have been directly linked to non-syndromic intellectual disability:

• X:2 balanced translocation leading to TM4SF2 inactivation .

• Premature stop codon TGA (gly218-to-ter): This mutation results in a truncated protein lacking the fourth transmembrane domain and cytoplasmic C-terminal tail .

• 2-bp deletion (564delGT): This mutation causes a frameshift leading to a premature stop codon at position 192, also resulting in a truncated protein lacking the fourth transmembrane domain and C-terminal tail .

The functional consequences of these mutations likely involve impaired dendritic spine formation and stability, as well as abnormal AMPA receptor trafficking, leading to defective synaptic transmission and plasticity. Research suggests that TSPAN7-related intellectual disability occurs because TSPAN7 loss results in alteration of dendritic filopodia and subsequent impaired cognitive functions .

What experimental approaches are most effective for studying TSPAN7's role in synaptic plasticity?

The most effective experimental approaches for studying TSPAN7's role in synaptic plasticity include:

• Primary neuronal culture: Hippocampal neurons cultured from embryonic rats provide an excellent model system for studying TSPAN7's effects on dendritic spine morphology and synaptic function .

• Molecular manipulation techniques:

  • RNA interference (RNAi) using shRNAs for specific knockdown of TSPAN7

  • Overexpression of wild-type and mutant TSPAN7 constructs

  • CRISPR/Cas9-mediated gene editing for precise manipulation of the endogenous TSPAN7 gene

• Imaging techniques:

  • Confocal microscopy for visualization of dendritic spines and filopodia

  • Super-resolution microscopy for detailed examination of TSPAN7 localization within dendritic spines

  • Live-cell imaging to track dynamic changes in spine morphology

• Electrophysiological recordings:

  • Patch-clamp recordings to measure synaptic transmission

  • Long-term potentiation (LTP) and long-term depression (LTD) protocols to assess synaptic plasticity

• Protein interaction studies:

  • Co-immunoprecipitation to confirm protein-protein interactions

  • Proximity ligation assays to detect interactions in situ

  • Fluorescence resonance energy transfer (FRET) to examine dynamic interactions

How is TSPAN7 expression altered in different cancer types, and what are the clinical correlations?

TSPAN7 expression shows distinct patterns across different cancer types with significant clinical correlations:

This differential expression pattern across cancer types suggests context-dependent roles of TSPAN7 in cancer progression, warranting tissue-specific research approaches.

What mechanisms underlie TSPAN7's effects on cancer cell proliferation and metastasis?

TSPAN7 influences cancer cell proliferation and metastasis through several mechanisms:

• Epithelial-Mesenchymal Transition (EMT) regulation:

  • Overexpression of TSPAN7 in NSCLC cells increases N-cadherin and vimentin expression while reducing E-cadherin expression

  • This shift in cadherin expression promotes the EMT process, enhancing cell migration and invasion potential

• Cell proliferation pathways:

  • TSPAN7 overexpression promotes cell growth in vitro, as demonstrated by cell proliferation assays and colony formation assays

  • Knockdown of TSPAN7 reduces lung cancer cell growth rate and colony formation ability

• In vivo tumor growth:

  • In xenograft models, TSPAN7 overexpression significantly increases tumor volume and accelerates growth of NSCLC cells

  • Tumors overexpressing TSPAN7 show molecular changes consistent with EMT (reduced E-cadherin, increased N-cadherin)

Methodologically, these findings have been established using multiple complementary approaches, including cell proliferation assays (CCK-8/MTT), colony formation assays, cell migration assays, Western blotting for EMT markers, and nude mice xenograft models .

What experimental design is optimal for investigating TSPAN7 as a potential therapeutic target in cancer?

An optimal experimental design for investigating TSPAN7 as a therapeutic target in cancer should include:

• Target validation studies:

  • Multi-cancer tissue microarray analysis to determine cancer-specific expression patterns

  • Kaplan-Meier survival analysis to correlate TSPAN7 expression with patient outcomes

  • CRISPR/Cas9 knockout or siRNA knockdown to validate dependency in cancer cell lines

• Mechanism elucidation:

  • RNA-seq and proteomics after TSPAN7 manipulation to identify downstream effectors

  • Chromatin immunoprecipitation (ChIP) to identify transcriptional regulators of TSPAN7

  • Co-immunoprecipitation and proximity labeling to identify cancer-specific protein interactions

• Therapeutic development pipeline:

  • High-throughput screening for small molecule inhibitors of TSPAN7-protein interactions

  • Monoclonal antibody development targeting TSPAN7's large extracellular loop

  • Testing of identified therapeutic candidates in:

    • Cell line panels representing multiple cancer types

    • 3D organoid cultures

    • Patient-derived xenograft models

• Biomarker development:

  • Identification of patient subgroups likely to respond to TSPAN7-targeted therapy

  • Development of companion diagnostics for patient stratification

  • Liquid biopsy approaches to monitor treatment response

• Combination therapy assessment:

  • Testing TSPAN7-targeted therapies in combination with standard chemotherapies

  • Evaluating synergy with immunotherapies and targeted therapies

This comprehensive approach ensures rigorous validation of TSPAN7 as a therapeutic target while developing clinically relevant strategies for intervention.

What are the most effective protein expression systems for producing recombinant TSPAN7 for structural studies?

Producing functional recombinant TSPAN7 for structural studies presents significant challenges due to its multiple transmembrane domains. The most effective protein expression systems include:

• Mammalian expression systems:

  • HEK293 and CHO cells provide proper folding and post-translational modifications

  • Inducible expression systems (e.g., tetracycline-inducible) offer controlled expression

  • Fusion tags such as GFP or His-tag facilitate purification while monitoring expression

• Insect cell expression systems:

  • Baculovirus-infected Sf9 or Hi5 cells often yield higher amounts of functional membrane proteins

  • The system supports proper folding of complex membrane proteins like TSPAN7

• Cell-free expression systems:

  • Wheat germ extract or E. coli-based cell-free systems supplemented with lipids or detergents

  • These systems allow direct incorporation into nanodiscs or liposomes during synthesis

• Yeast expression systems:

  • Pichia pastoris can produce large quantities of properly folded membrane proteins

  • The methylotrophic properties allow tight regulation of expression

For TSPAN7 specifically, mammalian and insect cell expression systems have proven most successful for producing properly folded protein. Purification strategies should employ mild detergents (DDM, LMNG) or native nanodiscs to maintain protein structure and function.

How can the membrane curvature sensitivity of TSPAN7 be quantitatively measured?

Based on the observation that TSPAN7 preferentially localizes to highly curved membrane structures like retraction fibers , several methodologies can be employed to quantitatively measure its membrane curvature sensitivity:

• Optical tweezers with Giant Plasma Membrane Vesicles (GPMVs):

  • Generate GPMVs from cells expressing TSPAN7-GFP

  • Use optical tweezers with streptavidin-coated beads to extract membrane tethers from biotin-PE-labeled GPMVs

  • Quantify the enrichment of TSPAN7-GFP on the highly curved tether compared to the less curved GPMV body

  • Use control membrane proteins (e.g., Pannexin1) that do not show curvature sensitivity for comparison

• Supported membrane tubes:

  • Create membrane tubes of defined diameters on microfabricated structures

  • Reconstitute purified TSPAN7 into these tubes

  • Measure protein density as a function of tube diameter

• Single-vesicle curvature assay:

  • Prepare vesicles of various sizes (30-400 nm diameter) with reconstituted TSPAN7

  • Use fluorescence correlation spectroscopy to measure protein density as a function of vesicle size

• Mathematical modeling:

  • Develop quantitative models relating TSPAN7 enrichment to membrane curvature

  • Parameters to measure include protein density ratio between high and low curvature regions and the critical curvature threshold for enrichment

This combination of approaches provides complementary quantitative data on TSPAN7's curvature sensing properties and the underlying biophysical mechanisms.

What imaging techniques are most suitable for visualizing TSPAN7 oligomerization and spiral formation?

Based on the discovery that TSPAN7 can polymerize into helical transmembrane skeletons , several advanced imaging techniques are particularly suitable for visualizing this process:

• Cryo-electron tomography:

  • Directly visualizes the spiral structure of TSPAN7 assemblies in cellular processes like retraction fibers

  • Preserves native structure through rapid freezing without chemical fixation

  • Enables 3D reconstruction at molecular resolution

  • Can be performed in situ on cellular samples, as demonstrated for wild-type TSPAN7 versus the T7 3M mutant

• Super-resolution microscopy:

  • STORM (Stochastic Optical Reconstruction Microscopy) provides resolution down to ~20 nm

  • PALM (Photoactivated Localization Microscopy) using photoactivatable TSPAN7 fusions

  • Structured Illumination Microscopy (SIM) for live-cell imaging of spiral formation dynamics

• Fluorescence fluctuation spectroscopy techniques:

  • Number and Brightness analysis to measure oligomerization state

  • Fluorescence Correlation Spectroscopy (FCS) to analyze diffusion properties

  • Photon Counting Histogram (PCH) to determine brightness distribution

• FRET-based approaches:

  • Homo-FRET measurements using polarization anisotropy

  • Spectral FRET imaging with differently colored TSPAN7 fusions

  • FLIM-FRET (Fluorescence Lifetime Imaging Microscopy) for quantitative assessment of protein proximity

Each technique offers complementary information, with cryo-electron tomography providing structural details at molecular resolution, super-resolution techniques enabling visualization in intact cells, and fluorescence fluctuation spectroscopy providing quantitative data on oligomerization kinetics.

What are the critical residues and domains in TSPAN7 that mediate its oligomerization and membrane curvature sensing?

Several critical residues and domains in TSPAN7 have been identified through comprehensive mutation analysis as essential for its oligomerization, spiral formation, and membrane curvature sensing:

• Key interaction interfaces:

  • E115 in the large extracellular loop (LEL)

  • Amino acids 190-199 (TVAATKVNQK) in the LEL

  • Amino acids 53-57 (ENSTN) in the small extracellular loop (SEL)

• Functional effects of mutations:

  • Single mutations (E115A, Δ53-57, or Δ190-199) each result in partial loss of TSPAN7 immobility

  • The triple mutant (T7 3M) shows almost complete loss of immobility

  • Cryo-tomography confirms absence of spiral structure in the T7 3M mutant

  • T7 3M shows significantly reduced enrichment on retraction fibers compared to wild-type TSPAN7

• Conservation patterns:

  • These interaction sites are fully conserved between mouse and human TSPAN7

  • E115 is conserved in the related protein TSPAN6 but not in TSPAN4

  • Neither aa53-57 nor aa190-199 are conserved in TSPAN6 or TSPAN4

These findings indicate that these specific residues form critical interaction interfaces that enable TSPAN7 to assemble into immobile spiral structures on curved membranes, a property that appears to be relatively unique among tetraspanin family members.

How does the polymerization of TSPAN7 into helical structures affect membrane properties and cellular functions?

The polymerization of TSPAN7 into helical transmembrane skeletons has significant effects on membrane properties and cellular functions:

• Membrane stabilization effects:

  • TSPAN7 spirals likely stabilize highly curved membrane structures such as retraction fibers and membrane tethers

  • In cells expressing wild-type TSPAN7, retraction fibers are significantly longer than in cells expressing the spiral-deficient T7 3M mutant

  • This suggests that TSPAN7 spirals provide structural support to maintain membrane tubules

• Influence on membrane curvature:

  • TSPAN7 shows strong preference for and enrichment on highly curved membrane surfaces

  • The spiral arrangement may either sense or induce membrane curvature

  • Cells expressing the triple mutant T7 3M form significantly shorter retraction fibers, suggesting TSPAN7 spirals may actively promote tubule extension

• Functional significance:

  • In neurons, this property may be critical for stabilizing dendritic filopodia during development

  • The ability to form structured oligomers may influence TSPAN7's interactions with binding partners like AMPA receptors, β1-integrin, and PICK1

  • In cancer, altered TSPAN7 expression may affect cell migration through changes in membrane dynamics and stability

These findings reveal a novel structural role for a tetraspanin protein, suggesting that TSPAN7's ability to form organized helical arrays represents a specialized adaptation that directly influences cellular morphology and function.

What is the relationship between TSPAN7's membrane organization and its role in AMPA receptor trafficking?

The relationship between TSPAN7's membrane organization and its role in AMPA receptor trafficking represents an intriguing intersection of its structural and functional properties:

• Spatial organization at synapses:

  • TSPAN7 localizes at excitatory synapses in cultured embryonic rat neurons

  • Its ability to form organized structures may create specialized membrane microdomains that facilitate receptor trafficking

• Molecular interactions:

  • TSPAN7 directly interacts with the PDZ domain of PICK1

  • PICK1 is a known regulator of AMPA receptor trafficking and the number of synaptic AMPA receptors

  • TSPAN7 associates with AMPA receptor subunit GluA2

  • TSPAN7 regulates PICK1 and GluA2/3 association

• Hypothesized mechanisms:

  • TSPAN7's spiral organization may create specialized membrane platforms that facilitate AMPA receptor endocytosis and exocytosis

  • The membrane curvature induced or stabilized by TSPAN7 may influence clathrin-mediated endocytosis of AMPA receptors

  • TSPAN7 spirals might organize multiple PICK1 molecules to efficiently capture and recycle AMPA receptors

• Experimental approach to test this relationship:

  • Super-resolution imaging of TSPAN7 and AMPA receptor subunits during synaptic plasticity

  • Comparison of AMPA receptor trafficking in cells expressing wild-type TSPAN7 versus spiral-deficient mutants

  • Correlation of TSPAN7 spiral formation with AMPA receptor dynamics using live-cell imaging