LAMTOR1 Antibody

Basic Research Questions

• What is LAMTOR1 and why is it significant in lysosomal research?

  LAMTOR1 (Late Endosome/Lysosome-Associated Membrane Protein 1) is a 161 amino acid membrane protein that specifically localizes to the surface of late endosomes/lysosomes. It functions as a key component of the lysosomal Ragulator complex, critically anchoring the complex to lysosomal membranes .

  LAMTOR1's significance stems from its multifunctional roles in:

  • Lysosomal positioning and trafficking

  • mTORC1 signaling pathway regulation

  • Calcium signaling through TRPML1 channel interaction

  • Immune regulation and antigen presentation

  • Metabolic homeostasis

  These diverse functions make LAMTOR1 a critical target for studying fundamental cellular processes like autophagy, endocytosis, nutrient sensing, and immune responses.

• What are the common applications of LAMTOR1 antibodies in research?

  LAMTOR1 antibodies serve multiple research applications with specific optimization parameters:

  Application	Recommended Dilution	Key Considerations
  Western Blot (WB)	1:5000-1:50000 or 1-2 μg/ml	Detects ~18 kDa band
  Immunofluorescence (IF)/ICC	1:50-1:500 or 20 μg/ml	Best for subcellular localization studies
  Immunohistochemistry (IHC)	Starting at 5 μg/ml	Tissue-specific optimization required
  Co-immunoprecipitation (Co-IP)	Application-dependent	Used for protein interaction studies

  These applications have been validated in multiple cell lines including A431, HEK-293, HeLa, HepG2, MCF-7, and U-87 MG cells , making LAMTOR1 antibodies versatile tools for diverse experimental systems.

• How does LAMTOR1 interact with the lysosomal trafficking machinery?

  LAMTOR1 regulates lysosomal trafficking through several mechanisms:

  • In neurons, LAMTOR1 knockdown significantly increases lysosomal motility in dendrites, enhancing both anterograde and retrograde trafficking

  • This regulation occurs through direct interaction with TRPML1 calcium channels, where LAMTOR1's N-terminal domain (particularly residues 20-60) is critical for inhibiting TRPML1-mediated calcium release

  • LAMTOR1 affects lysosomal positioning, as its knockdown causes accumulation of lysosomes in proximal dendrites of hippocampal neurons

  • The effect on lysosomal trafficking appears independent of mTORC1 activity, as Raptor knockdown or Torin 1 treatment does not replicate these effects

  These interactions position LAMTOR1 as a key regulator of lysosomal dynamics in specialized cellular contexts.

Intermediate Research Questions

• What methodological approaches can be used to study LAMTOR1's role in lysosomal positioning?

  Several validated methodological approaches can be employed:

  1. Lysosomal tracking:

     • Live-cell imaging with LysoTracker labeling

     • Analysis of mobile versus static lysosomes using kymographs

     • Quantification of directional movement (anterograde vs. retrograde)

  2. Genetic manipulation:

     • AAV-mediated shRNA knockdown (typically analyzed 14 days post-transduction)

     • Expression of shRNA-resistant LAMTOR1 constructs for rescue experiments

     • Generation of deletion constructs targeting specific domains (N-terminal vs. C-terminal)

  3. Immunofluorescence analysis:

     • Colocalization studies with lysosomal markers (LAMP2, cathepsin B)

     • Measurement of lysosomal density in specific cellular compartments

     • Comparative analysis with other endosomal markers (EEA1, Rab11)

  4. Pharmacological interventions:

     • GPN (glycyl-L-phenylalanine 2-naphthylamide) for lysosomal osmotic lysis

     • mTOR inhibitors like Torin 1 to distinguish mTORC1-dependent effects

     • TRPML1 inhibitors like ML-SI1 for pathway validation

• How can researchers verify LAMTOR1 antibody specificity in their experimental systems?

  Verifying antibody specificity is critical for reliable results. Recommended approaches include:

  1. Positive controls:

     • Test in validated cell lines (A431, HEK-293, HeLa, HepG2, MCF-7)

     • Compare results with published molecular weight (18 kDa)

     • Verify subcellular localization to lysosomes with co-staining

  2. Negative controls:

     • LAMTOR1 knockdown using validated shRNA constructs

     • CRISPR/Cas9-generated knockout cell lines

     • Secondary antibody-only controls

  3. Specificity tests:

     • Peptide competition assays

     • Isotype controls

     • Cross-validation using antibodies recognizing different epitopes

  4. Rescue experiments:

     • Re-expression of LAMTOR1 in knockdown/knockout systems

     • Testing phenotype reversal with shRNA-resistant constructs

  Antibody validation should be performed for each experimental application (WB, IF, IHC) separately, as specificity may vary across techniques.

• What is the functional relationship between LAMTOR1 and TRPML1, and how can it be studied?

  LAMTOR1 directly interacts with and inhibits TRPML1, a lysosomal calcium channel, through:

  1. Interaction mechanisms:

     • LAMTOR1's N-terminal domain (residues 20-60, particularly 20-31) binds directly to TRPML1

     • This interaction inhibits TRPML1-mediated lysosomal calcium release

  2. Experimental approaches to study this relationship:

     • Co-immunoprecipitation of LAMTOR1 and TRPML1

     • Domain mapping using deletion constructs (ΔN20-60, ΔN20-31, ΔN42-60, ΔC144-161)

     • Calcium imaging to measure TRPML1 activity

     • Lysosomal trafficking analysis with TRPML1 inhibitors (ML-SI1)

  3. Functional consequences:

     • LAMTOR1 knockdown enhances lysosomal trafficking through increased TRPML1 activity

     • This pathway affects lysosomal positioning in proximal dendrites

     • These effects impact neuronal plasticity (LTP induction, LTD expression)

  Understanding this interaction has significant implications for neuronal function and may provide insights into neurological disorders associated with lysosomal dysfunction.

Advanced Research Questions

• How can LAMTOR1 antibodies be utilized to investigate its role in mTORC1 signaling pathway regulation?

  LAMTOR1 antibodies can elucidate the complex relationship between LAMTOR1 and mTORC1 through:

  1. Subcellular localization studies:

     • Immunofluorescence co-localization of LAMTOR1 with mTOR and Rag GTPases on lysosomes

     • Analysis of mTOR translocation to lysosomes in LAMTOR1-manipulated cells

     • Live-cell imaging with fluorescently-tagged LAMTOR1 and mTORC1 components

  2. Signaling pathway analysis:

     • Western blot assessment of mTORC1 downstream targets (phospho-S6K1, phospho-S6) in LAMTOR1 knockdown/knockout systems

     • Comparison with Raptor knockdown effects to distinguish direct LAMTOR1 effects

     • Analysis under different nutrient conditions to evaluate amino acid sensing

  3. Protein interaction mapping:

     • Co-immunoprecipitation of LAMTOR1 with Ragulator components and Rag GTPases

     • Proximity labeling approaches (BioID, APEX) to identify interaction networks

     • Domain-specific mutations to disrupt specific interactions

  4. Tissue-specific analyses:

     • Comparison of LAMTOR1-mTORC1 relationships across cell types (neurons, macrophages, hepatocytes)

     • Assessment of differential responses to nutrient availability

  Research indicates that while LAMTOR1 affects mTORC1 signaling, its role in lysosomal positioning appears independent of mTORC1 activity, suggesting context-dependent functions .

• What methodologies can be employed to investigate LAMTOR1's role in immune regulation and MHC-II expression?

  Recent research has revealed LAMTOR1's critical role in immune regulation, which can be studied through:

  1. Endocytic pathway analysis:

     • Flow cytometry measurement of cell surface MHC-II levels in LAMTOR1 knockdown cells

     • Tracking MHC-II internalization and degradation using antibody feeding assays

     • Co-localization studies of MHC-II with endosomal/lysosomal markers

  2. Autophagy-endocytosis coupling:

     • Analysis of amphisome formation through co-localization of:

       • LC3B (autophagosome marker) with CD63/TSG101 (endosome markers)

       • LC3B/CD63 with LAMP1 (lysosome marker)

     • Transmission electron microscopy to visualize autophagosomes and amphisomes

  3. T cell response assessment:

     • Co-culture experiments with CD8+ and CD4+ T cells

     • Measurement of tumor cell apoptosis in response to T cell killing

     • Analysis of T cell infiltration in tissue samples

  4. Mechanistic dissection:

     • Combinatorial knockdown approaches (LAMTOR1 + RAB7, LAMTOR1 + ATG7)

     • In vivo tumor studies with genetic manipulation of LAMTOR1 and endocytic/autophagic pathway components

  These approaches have revealed that LAMTOR1 downregulates MHC-II surface expression through endocytic degradation, facilitating immune escape in hepatocellular carcinoma by reducing CD4+ T cell recognition and subsequent CD8+ T cell killing .

• How can researchers design experiments to differentiate between LAMTOR1's direct effects and indirect consequences through the Ragulator complex?

  Distinguishing direct LAMTOR1 functions from those mediated through the Ragulator complex requires sophisticated experimental design:

  1. Domain-specific manipulations:

     • Expression of LAMTOR1 mutants that selectively disrupt interaction with:

       • TRPML1 (N-terminal deletions, particularly residues 20-31)

       • Other Ragulator components (LAMTOR2-5)

       • Membrane anchoring domains

     • Comparison with complete LAMTOR1 knockdown phenotypes

  2. Comparative manipulations of Ragulator components:

     • Knockdown of LAMTOR1 versus LAMTOR2-5

     • Analysis of compensatory effects when individual components are depleted

     • Studies have shown LAMTOR2 knockdown leads to down-regulation of LAMTOR4 and LAMTOR1 , indicating complex interdependencies

  3. Pathway-specific readouts:

     • mTORC1 activity (phospho-S6K1, phospho-S6)

     • Lysosomal positioning and trafficking

     • TRPML1-mediated calcium release

     • MHC-II surface expression and endocytosis

  4. Temporal manipulation:

     • Acute versus chronic LAMTOR1 depletion

     • Inducible knockdown/knockout systems

     • Time-course analyses to distinguish primary from secondary effects

  Research has demonstrated that certain LAMTOR1 functions (like lysosomal trafficking regulation) appear independent of mTORC1 activity, suggesting direct mechanisms beyond its role in the Ragulator complex .

• What approaches can be used to study tissue-specific functions of LAMTOR1 in metabolic regulation and disease models?

  LAMTOR1's tissue-specific roles, particularly in metabolism and disease, can be investigated through:

  1. Cell-type specific genetic manipulations:

     • Conditional knockout models (e.g., myeloid-specific LAMTOR1 knockout)

     • AAV-mediated tissue-specific knockdown

     • CRISPR/Cas9 tissue-specific gene editing

  2. Metabolic phenotyping:

     • Dietary challenge experiments (high-fat diet) in tissue-specific knockout models

     • Assessment of obesity development and insulin sensitivity

     • Analysis of liver-specific effects on Kupffer cells and immune cell populations

  3. Cancer models and immune escape mechanisms:

     • Tumor xenograft studies with LAMTOR1 manipulation

     • Analysis of:

       • MHC-II surface expression

       • CD4+ and CD8+ T cell infiltration

       • Tumor growth and immunogenicity

     • Combined targeting of endocytic and autophagic pathways (LAMTOR1 + RAB7, LAMTOR1 + ATG7)

  4. Mechanistic analyses in specific tissues:

     • Flow cytometry to quantify immune cell populations (CD45+, F4/80+CD11b-, F4/80-CD11b+)

     • Assessment of macrophage polarization (M1/M2 markers)

     • Tissue-specific protein interaction studies

  Research has demonstrated that myeloid-specific LAMTOR1 knockout prevents dietary obesity and metabolic disorders through effects on liver Kupffer cells , while in hepatocellular carcinoma, LAMTOR1 promotes immune escape by reducing MHC-II surface expression .

Additional Considerations for Researchers

• What are the key experimental controls necessary when studying LAMTOR1 knockdown effects?

  Robust experimental design for LAMTOR1 studies requires:

  1. Genetic manipulation controls:

     • Scrambled shRNA controls for knockdown experiments

     • Empty vector controls for overexpression studies

     • shRNA-resistant LAMTOR1 rescue constructs to confirm specificity

     • CRISPR/Cas9 controls with non-targeting gRNAs

  2. Functional pathway controls:

     • Parallel knockdown of related components (e.g., LAMTOR2, Raptor)

     • Pharmacological inhibitors as complementary approaches:

       • mTOR inhibitors (Torin 1)

       • TRPML1 inhibitors (ML-SI1)

       • Myosin II inhibitors (blebbistatin)

     • Domain-specific mutants to pinpoint interaction regions

  3. Cell biological controls:

     • Analysis of multiple organelle markers to confirm specificity (EEA1, Rab11)

     • Assessment of cell viability and general cellular architecture

     • Time-course studies to distinguish primary from secondary effects

  4. Tissue/cell type considerations:

     • Validation across multiple cell types when possible

     • Consideration of tissue-specific LAMTOR1 functions

     • Wild-type comparisons for in vivo models

  These controls have been essential in establishing LAMTOR1's specific roles in lysosomal trafficking, positioning, and immune regulation that are distinct from its general functions in the Ragulator complex .

• How can researchers resolve contradictory findings about LAMTOR1 functions in different experimental systems?

  Addressing contradictory results requires systematic analysis:

  1. Context-dependent functions:

     • LAMTOR1 exhibits distinct functions across different cell types:

       • In neurons: regulates lysosomal trafficking and positioning

       • In macrophages: affects migration and metabolic regulation

       • In cancer cells: influences MHC-II expression and immune escape

     • Experimental design should account for these tissue-specific roles

  2. Pathway interconnections:

     • LAMTOR1 participates in multiple interacting pathways:

       • Ragulator complex and mTORC1 signaling

       • TRPML1-mediated calcium signaling

       • Endocytic and autophagic pathways

     • Changes in one pathway may have compensatory effects in others

  3. Methodological reconciliation approaches:

     • Direct comparative studies using identical:

       • Genetic manipulation methods (shRNA sequences, expression systems)

       • Timeframes (acute vs. chronic effects)

       • Readout assays

     • Comprehensive pathway analysis rather than isolated endpoints

     • Multi-omics approaches to capture system-wide effects

  4. Molecular dissection strategies:

     • Domain-specific mutations to separate different LAMTOR1 functions

     • Temporal control of LAMTOR1 manipulation (inducible systems)

     • Combined in vitro and in vivo validation

  Research has shown that while LAMTOR1 broadly functions in the Ragulator complex, its specific effects can vary dramatically by context - some functions (like lysosomal trafficking regulation) appear independent of mTORC1 activity , while others (like immune regulation) involve complex interplay between endocytosis and autophagy .