CLTB Human

What is CLTB and what is its primary function in human cells?

CLTB (Clathrin Light Chain B) is a member of the clathrin light chain family that plays a critical role in the formation of coated vesicles. It functions as a key structural component of the lattice-type cytoplasmic face of coated pits and vesicles which capture macromolecules during receptor-mediated endocytosis. CLTB forms a triskelion structure comprised of 3 clathrin heavy chains and 3 light chains, which interact to form a polyhedral pattern encircling vesicles. This structure is essential for clathrin-mediated endocytosis (CME), which controls numerous cellular physiological processes including the internalization of growth factors and receptors, pathogen entry, and synaptic transmission .

How does CLTB differ structurally from CLTA?

While both CLTB and CLTA are clathrin light chains, they are encoded by different genes located on different chromosomes. CLTB is encoded by a gene located on chromosome 4 at position 4q2-q3, whereas CLTA is encoded by a gene on chromosome 12 at position 12q23-q24 . The primary structural difference between the two involves their neuron-specific insertions: CLTB's insertion is encoded by a single exon, while CLTA's insertion is encoded by two exons. The first of CLTA's neuron-specific exons shows homology to the corresponding CLTB exon. Additionally, an intronic sequence of the CLTB gene bears similarity to the second neuron-specific exon of the CLTA gene .

What are the physical characteristics of recombinant CLTB Human protein?

Recombinant CLTB Human produced in E. coli is a single, non-glycosylated polypeptide chain containing 234 amino acids (1-211 a.a. of the native protein plus a 23 amino acid His-tag at the N-terminus). It has a molecular mass of 25.6kDa, though it may appear larger on SDS-PAGE. The purified protein typically appears as a sterile filtered colorless solution and is formulated in a buffer containing 20mM Tris-HCl (pH 8.0), 10% glycerol, 0.2mM PMSF, and 100mM NaCl at a concentration of 1mg/ml .

How is the CLTB gene organized and what splicing variations exist?

The CLTB gene in humans maps to the long arm of chromosome 4 at position 4q2-q3. Research has shown that the neuron-specific insertions in CLTB protein are encoded by discrete exons, confirming that clathrin light chains undergo alternative mRNA splicing to generate tissue-specific protein isoforms. Specifically, the insertion sequence of CLTB is encoded by a single exon, unlike CLTA which requires two exons for its neuron-specific insertion . This alternative splicing is a critical mechanism that allows for the generation of tissue-specific variants of CLTB with potentially different functional properties.

What methodologies are most effective for studying CLTB expression patterns across different tissues?

For comprehensive analysis of CLTB expression patterns, researchers should consider a multi-modal approach:

• RNA-Seq and qRT-PCR: To quantify tissue-specific expression levels and identify splice variants

• Western blotting: For protein-level validation using isoform-specific antibodies

• Immunohistochemistry: To visualize spatial distribution in different tissues

• Single-cell RNA sequencing: To reveal cell-type specific expression patterns

• Fluorescence in situ hybridization (FISH): For visualizing mRNA localization

When studying neuron-specific isoforms, researchers should include appropriate controls and validate findings across multiple methodologies, as expression levels may vary significantly between tissues. Particular attention should be paid to the neuron-specific exon of CLTB, as its inclusion/exclusion represents a key regulatory mechanism for tissue-specific function .

How do mutations in CLTB affect clathrin-mediated endocytosis at the molecular level?

Mutations in CLTB can disrupt clathrin-mediated endocytosis (CME) through several mechanisms:

• Triskelion assembly disruption: Mutations affecting the interaction domains can prevent proper formation of the characteristic three-legged structure

• Altered membrane recruitment: Changes in binding affinity to adaptor proteins can reduce vesicle formation efficiency

• Vesicle size regulation: Some mutations alter the geometry of the clathrin lattice, resulting in abnormal vesicle morphology

• Cargo selectivity changes: Structural alterations can modify interactions with specific cargo adaptors

Methodologically, these effects can be studied using reconstitution assays with purified components, live-cell imaging with fluorescently tagged CLTB variants, and electron microscopy to visualize structural abnormalities. CRISPR-Cas9 gene editing provides a powerful approach for introducing specific mutations and studying their functional consequences in cellular contexts.

What are the optimal conditions for storing and handling recombinant CLTB protein for experimental use?

For optimal stability and activity of recombinant CLTB protein:

• Short-term storage (2-4 weeks): Store at 4°C

• Long-term storage: Store frozen at -20°C

• For extended storage periods, add a carrier protein (0.1% HSA or BSA) to enhance stability

• Avoid multiple freeze-thaw cycles as they can cause protein denaturation and activity loss

• The protein is typically supplied at 1mg/ml in a buffer containing 20mM Tris-HCl (pH 8.0), 10% glycerol, 0.2mM PMSF, and 100mM NaCl

When designing experiments, researchers should include appropriate controls to ensure the protein maintains its expected activity after storage and handling procedures.

How can researchers effectively study the interactions between CLTB and other components of the clathrin triskelion?

To study CLTB interactions within the clathrin triskelion complex:

• Pull-down assays and co-immunoprecipitation: These techniques can identify direct binding partners of CLTB

• Surface plasmon resonance (SPR): For measuring binding kinetics and affinity constants

• Fluorescence resonance energy transfer (FRET): To visualize protein-protein interactions in living cells

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): For mapping interaction interfaces at high resolution

• Cryo-electron microscopy: To determine the structural arrangement of CLTB within the assembled triskelion

When designing these experiments, it's critical to consider the triskelion structure comprising 3 clathrin heavy chains and 3 light chains, and how mutations or modifications might affect the polyhedral pattern formation that encircles vesicles during endocytosis .

What are the challenges and solutions in producing high-purity CLTB for structural studies?

The production of high-purity CLTB for structural studies presents several challenges:

• Expression system selection:

  • Challenge: E. coli expression systems may yield non-glycosylated protein that differs from the native form

  • Solution: Use mammalian expression systems for studies requiring post-translational modifications, or E. coli for basic structural work (as described in the product specification )

• Protein solubility:

  • Challenge: CLTB may form inclusion bodies when overexpressed

  • Solution: Optimize expression conditions (temperature, induction time) or use solubility-enhancing tags

• Purification complexity:

  • Challenge: Obtaining >95% purity required for crystallography

  • Solution: Multi-step purification strategy combining His-tag affinity chromatography with ion exchange and size exclusion chromatography

• Stability during concentration:

  • Challenge: Protein aggregation during concentration steps

  • Solution: Include stabilizing agents like glycerol (10%) as used in the commercial formulation

• Crystal formation:

  • Challenge: Obtaining diffraction-quality crystals

  • Solution: Screen various crystallization conditions and consider protein engineering to remove flexible regions

How do tissue-specific isoforms of CLTB contribute to specialized endocytic functions?

Tissue-specific isoforms of CLTB, generated through alternative splicing, contribute to specialized endocytic functions in different cell types. In neurons, the inclusion of the neuron-specific exon (as identified in genomic studies ) alters the protein's properties to support the high-rate endocytosis required during synaptic vesicle recycling.

Research methodologies to investigate these specialized functions include:

• Isoform-specific knockdown/knockout experiments

• Rescue experiments with different CLTB isoforms

• Live-cell imaging of fluorescently tagged isoforms

• Electrophysiological measurements in neurons expressing different CLTB variants

• Quantitative endocytosis assays comparing the efficiency of different isoforms

These approaches have revealed that neuron-specific CLTB isoforms support the rapid kinetics of synaptic vesicle recycling, while other tissues express variants optimized for their specific endocytic requirements.

What role does CLTB play in pathological conditions and potential therapeutic applications?

CLTB's critical role in clathrin-mediated endocytosis (CME) links it to several pathological conditions:

• Neurological disorders:

  • CME dysfunction affects synaptic transmission

  • Alterations in CLTB function may contribute to neurodegenerative processes

• Cancer progression:

  • Dysregulated receptor endocytosis can enhance growth factor signaling

  • CLTB may influence the internalization and recycling of receptors that drive tumor growth

• Viral and bacterial infections:

  • Many pathogens exploit CME for cellular entry

  • CLTB functionality affects infection efficiency of certain pathogens

Therapeutic approaches targeting CLTB or its interactions include:

• Small molecule modulators of clathrin assembly/disassembly

• Peptide inhibitors targeting specific CLTB interaction surfaces

• Gene therapy approaches to restore normal CLTB function in tissues with defective expression

When designing experiments to investigate these therapeutic applications, researchers should consider tissue-specific isoforms and their differential roles in normal and pathological conditions.

How does CLTB interact with other endocytic proteins to regulate clathrin-mediated endocytosis?

CLTB interacts with numerous proteins to regulate clathrin-mediated endocytosis through a complex network of interactions:

• Adaptor protein complexes (AP-1, AP-2):

  • CLTB binding to these complexes facilitates cargo selection and coat assembly

  • Experimental approach: In vitro binding assays with purified components and structural analysis

• Auxilin and HSC70:

  • These proteins interact with CLTB during uncoating of clathrin-coated vesicles

  • Methodology: ATP hydrolysis assays and live-cell imaging of uncoating kinetics

• Hip1R and other actin-binding proteins:

  • CLTB mediates connections to the cytoskeleton

  • Research technique: Co-localization studies using super-resolution microscopy

• Regulatory kinases:

  • Phosphorylation of CLTB modulates its binding properties

  • Approach: Phosphoproteomic analysis and mutational studies of phosphorylation sites

Understanding these interactions requires combined approaches including biochemical assays, advanced imaging techniques, and systems biology methods to integrate the complex data into coherent mechanistic models of endocytosis regulation.

What are the best approaches to study CLTB dynamics in living cells?

To effectively study CLTB dynamics in living cells, researchers should consider these advanced methodological approaches:

• Fluorescent protein tagging:

  • Construct CLTB-GFP/mCherry fusion proteins, ensuring the tag doesn't interfere with function

  • Validate proper localization and function compared to endogenous protein

• Live-cell imaging techniques:

  • Total Internal Reflection Fluorescence (TIRF) microscopy: For visualizing events at the plasma membrane

  • Spinning disk confocal microscopy: For rapid 3D imaging with reduced photobleaching

  • Super-resolution techniques (STORM, PALM): For nanoscale resolution of clathrin structures

• Quantitative analysis approaches:

  • Single particle tracking: To follow individual clathrin-coated pits

  • Fluorescence Recovery After Photobleaching (FRAP): To measure CLTB turnover rates

  • Automated image analysis: For high-throughput quantification of dynamics

• Optogenetic approaches:

  • Light-inducible dimerization systems to perturb CLTB interactions

  • Local activation of signaling pathways that regulate endocytosis

When implementing these techniques, researchers should carefully control for expression levels of tagged proteins, as overexpression may alter the normal dynamics of clathrin-mediated endocytosis.

How can researchers accurately distinguish between the functions of CLTB and CLTA in experimental systems?

Distinguishing between CLTB and CLTA functions requires sophisticated experimental designs:

• Isoform-specific genetic manipulation:

  • CRISPR-Cas9 knockout of individual genes (CLTB on chromosome 4, CLTA on chromosome 12 )

  • siRNA with validated specificity for each isoform

  • Rescue experiments with one isoform in cells lacking both

• Domain-specific antibodies and probes:

  • Develop antibodies targeting unique epitopes of each protein

  • Create isoform-specific fluorescent probes for live imaging

• Tissue and cell-type comparative analysis:

  • Leverage natural variation in CLTA:CLTB ratios across tissues

  • Analyze phenotypes in tissues where one isoform predominates

• Biochemical approach:

  • In vitro reconstitution with purified components

  • Compare vesicle formation efficiency and properties with CLTB vs. CLTA

• Computational modeling:

  • Molecular dynamics simulations to identify isoform-specific interaction patterns

  • Systems biology approaches to model differential network effects

The ultimate experimental strategy should combine multiple approaches to build a comprehensive understanding of the distinct roles of these related but functionally distinct proteins.

What computational approaches are most effective for predicting CLTB interactions and functional sites?

Modern computational approaches offer powerful tools for predicting CLTB interactions and functional sites:

• Structural prediction and analysis:

  • AlphaFold2 and RoseTTAFold: For high-accuracy prediction of CLTB structure

  • Molecular dynamics simulations: To identify flexible regions and stable interaction interfaces

  • Docking algorithms: To predict protein-protein interactions with binding partners

• Machine learning approaches:

  • Neural networks trained on interaction databases to predict novel binding partners

  • Feature extraction from amino acid sequences to identify functional motifs

• Network-based methods:

  • Interactome mapping to identify hub proteins connected to CLTB

  • Pathway enrichment analysis to predict biological processes involving CLTB

• Evolution-based prediction:

  • Conservation analysis to identify functionally important residues

  • Coevolution analysis to detect residue pairs involved in interactions

• Integrative approaches:

  • Combining experimental data (crosslinking-MS, HDX-MS) with computational predictions

  • Multi-scale modeling from atomic to cellular levels

These computational methods should be validated through targeted experimental approaches, creating an iterative cycle between prediction and verification that can accelerate the discovery of functional sites and interactions.