MEMO1 Human

What is the molecular structure of human MEMO1 and how does it relate to its function?

MEMO1's structure reveals striking similarity to iron-containing extradiol dioxygenases, with a comparable structural fold and iron coordination mode. The protein structure solved in complex with iron and copper demonstrates specific metal-binding pockets that influence its biological activity. This structural arrangement enables MEMO1 to bind iron with high affinity under redox conditions mimicking intracellular environments, suggesting potential enzymatic functions in cellular metabolism . The structural homology to dioxygenases indicates MEMO1 may be involved in the biosynthesis or processing of signaling molecules within cells .

What are the primary cellular functions of MEMO1 in humans?

MEMO1 functions across multiple cellular processes, primarily as a mediator of ERBB2 signaling that controls cell migration by relaying extracellular chemotactic signals to the microtubule cytoskeleton . It operates through the MEMO1-RHOA-DIAPH1 signaling pathway, playing a crucial role in ERBB2-dependent stabilization of microtubules at the cell cortex . MEMO1 regulates GSK3B activity to control the localization of APC and CLASP2 to the cell membrane, allowing MACF1 localization required for microtubule capture and stabilization . Additionally, MEMO1 functions as a metal-binding protein that modulates iron homeostasis in cancer cells and coordinates reduced copper ions to prevent ROS generation .

How is MEMO1 expression regulated in different human tissues?

MEMO1 expression varies across tissues, with notably elevated levels in multiple cancer types. Researchers investigating tissue-specific expression should employ tissue microarrays with immunohistochemistry using validated anti-MEMO1 antibodies. RNA sequencing and quantitative PCR methodologies can provide comprehensive transcriptomic profiles across normal and pathological tissue samples. For cancer contexts, particular attention should be paid to breast tissues, where MEMO1 overexpression correlates with metastatic potential and reduced patient survival . When analyzing expression data, normalization against appropriate housekeeping genes is essential, and verification through multiple methodological approaches strengthens the validity of tissue-specific expression patterns.

How does MEMO1 contribute to cancer metastasis mechanisms?

MEMO1 enhances metastatic potential primarily by modulating breast cancer cell migration and invasion. Methodologically, researchers should investigate this through multiple approaches: (1) In vitro migration assays using transwell chambers to quantify invasive capacity of cells with modulated MEMO1 expression; (2) Live cell imaging to visualize cytoskeletal dynamics and lamellipodia formation in response to MEMO1 activity; (3) Xenograft models to assess metastatic spread in vivo, comparing MEMO1 knockdown/knockout cells with controls . The relationship between MEMO1 and ERBB2 (HER2) receptor signaling is particularly important, as MEMO1 relays activation signals to the microtubule cytoskeleton, promoting lamellipodia growth and enabling cancer cell migration . Additionally, MEMO1 contributes to carcinogenesis through the insulin receptor substrate protein 1 pathway and interactions with extranuclear estrogen receptors .

What experimental approaches best assess the impact of MEMO1 on cancer cell survival?

To comprehensively assess MEMO1's impact on cancer cell survival, researchers should implement multiple complementary methodologies:

• CRISPR/Cas9-mediated knockout or knockdown strategies using synthetic crRNA technology targeting MEMO1 exons (similar to the approach using crRNAs targeting exons 4-6 as described in the literature)

• Validation of knockout efficiency through genomic cleavage detection and western blotting

• Proliferation assays using systems like Incucyte S3 to track cell confluency over 120-140 hours

• Analysis of cell cycle distribution using flow cytometry with propidium iodide staining

• Assessment of apoptotic markers through flow cytometry or western blot detection of cleaved caspase-3/7

• Metabolic viability assays (MTT, ATP-based) under various stress conditions including hypoxia, oxidative stress, and iron chelation

This multi-faceted approach will reveal both direct and context-dependent effects of MEMO1 on cancer cell survival across different microenvironmental conditions.

How can MEMO1 expression be therapeutically targeted in cancer treatment strategies?

Development of therapeutic approaches targeting MEMO1 requires understanding its genetic interactions and functional networks. Methodological approaches should include: (1) Small molecule library screening to identify compounds that disrupt MEMO1 binding to key partners such as ERBB2 or iron/copper; (2) Peptide mimetics designed to interfere with protein-protein interactions; (3) Exploration of synthetic lethality approaches targeting genes with strong genetic interactions with MEMO1, particularly those showing essentiality in MEMO1-high cancer cells like TFR2, SLC25A28, and ACO1 ; (4) Development of iron chelators specifically targeting MEMO1-dependent iron homeostasis pathways; (5) PROTAC (Proteolysis-targeting chimera) design to induce MEMO1 degradation. When evaluating therapeutic efficacy, researchers should assess effects on both primary tumor growth and metastatic potential, using both in vitro and in vivo models with genetically diverse cancer cell lines.

What methodologies best characterize MEMO1's interaction with iron and its role in iron homeostasis?

To characterize MEMO1-iron interactions, researchers should employ multiple complementary approaches:

• Biochemical analysis with purified MEMO1 protein to determine binding affinities under various redox conditions using isothermal titration calorimetry (ITC) or microscale thermophoresis

• Structural studies using X-ray crystallography to visualize iron coordination sites (similar to the studies that revealed structural similarity to iron-containing dioxygenases)

• Cellular iron distribution analysis using fluorescent iron probes or inductively coupled plasma mass spectrometry (ICP-MS) in MEMO1 knockout versus wild-type cells

• Assessment of mitochondrial iron levels using mitochondria-targeted iron sensors or fractionation followed by ICP-MS

• Investigation of iron-dependent proteins' activity (aconitases, iron-sulfur cluster proteins) in MEMO1-modulated cell lines

• Ferroptosis sensitivity assays comparing MEMO1-high versus MEMO1-low cells

These methodological approaches should be performed under both normal conditions and iron depletion (using chelators like DFX at subtoxic concentrations of around 1μM) to reveal MEMO1's specific role in maintaining cellular iron homeostasis .

How does MEMO1 coordinate copper ions and what are the functional implications?

MEMO1 specifically coordinates reduced copper ions (Cu(I)) with two Cu(I) ions bound per protein molecule . This binding shields the metal ions from participating in redox reactions that would generate reactive oxygen species (ROS) . Methodologically, researchers investigating this phenomenon should:

• Employ biophysical methods under both reducing and oxidizing conditions to assess copper binding specificity

• Utilize copper chelators and competitors to determine binding affinities

• Conduct site-directed mutagenesis of putative copper-binding residues to identify critical coordination sites

• Assess ROS generation using fluorescent probes (DCF-DA, MitoSOX) in cells with modulated MEMO1 expression

• Perform proximity-based assays (proximity ligation, FRET) to evaluate MEMO1 interaction with copper chaperones like Atox1

• Analyze cellular copper distribution using appropriate copper probes or ICP-MS in fractionated cell components

These approaches will help elucidate MEMO1's function as a Cu(I) chelator that potentially shuttles copper ions to Atox1 in the secretory pathway, thereby protecting cells from copper-mediated toxicity .

How do iron and copper binding by MEMO1 interact with each other and affect cellular redox balance?

This question requires sophisticated experimental approaches addressing the potential interplay between MEMO1's iron and copper binding functions. Methodological approaches should include:

• Competitive binding assays with purified MEMO1 to determine if iron and copper binding are mutually exclusive or cooperative

• Mass spectrometry analysis of MEMO1-metal complexes isolated from cells under various metal loading conditions

• Redox potential measurements of MEMO1 when bound to different metal ions

• ROS measurements in cells with MEMO1 variants selectively impaired in either iron or copper binding

• Assessment of mitochondrial function and morphology using respirometry, membrane potential measurements, and high-resolution microscopy when modulating cellular iron/copper availability

• Analysis of iron-sulfur cluster protein activities when manipulating MEMO1 and copper levels

These approaches can reveal whether MEMO1 functions primarily as an iron-binding protein that modulates cellular iron homeostasis, a copper chelator that prevents ROS generation, or integrates both functions in a context-dependent manner.

What is the evidence linking MEMO1 to autism spectrum disorders?

MEMO1 has been implicated in autism through genetic and functional studies. Methodologically, researchers should approach this question by:

• Analyzing MEMO1 variants in autism cohorts, particularly focusing on splice-site variants as identified by Iossifov et al. (2014)

• Conducting functional assays with autism-associated MEMO1 variants to assess their impact on radial glial cell morphology

• Developing mouse models with conditional Memo1 knockout to evaluate neurodevelopmental phenotypes

• Investigating the effects of MEMO1 deletion or knockdown on radial glial cell basal process hyperbranching and disrupted tiling

• Assessing the impact on radial unit assembly and neuronal layering in developing cerebral cortex

• Performing rescue experiments with wild-type MEMO1 compared to autism-associated variants

Nakagawa et al. (2019) demonstrated that Memo1 gene deletion or knockdown in mice resulted in hyperbranching of radial glial cell basal processes and disrupted RGC tiling, leading to aberrant radial unit assembly and neuronal layering . Furthermore, MEMO1 containing an autism-associated splice-site variant (c.143+1G>A) failed to rescue RG tiling defects in Memo1 conditional knockout mice, unlike wild-type MEMO1 .

What cellular mechanisms link MEMO1 dysfunction to abnormal cortical development?

To investigate the cellular mechanisms connecting MEMO1 to cortical development, researchers should employ:

• In utero electroporation to manipulate MEMO1 expression in developing rodent brains

• Time-lapse imaging of radial glial cell dynamics in brain slice cultures

• Analysis of cytoskeletal organization in radial glial cells with altered MEMO1 expression

• Assessment of cell-cell interactions and boundary formation between adjacent radial units

• Evaluation of neuronal migration patterns along radial processes

• Investigation of potential interactions between MEMO1 and other proteins involved in cortical development

These approaches can help determine how MEMO1 regulates radial glial cell morphology, particularly focusing on the tiling of basal processes and the formation of proper radial units essential for cortical development.

How can researchers best create and validate MEMO1 knockout and knockdown models?

For rigorous MEMO1 knockout/knockdown model development, researchers should follow this methodological framework:

• Design multiple CRISPR/Cas9 targeting strategies using synthetic crRNA technology targeting different MEMO1 exons (exons 4-6 have been successfully targeted)

• Verify genomic cleavage using detection kits that amplify the targeted region

• Generate both complete knockouts and partial knockdowns through careful clone selection

• Validate MEMO1 depletion at protein level through western blotting with multiple validated antibodies

• Perform functional validation through known MEMO1-dependent phenotypes (cell migration, iron homeostasis)

• Authenticate cell lines by short tandem repeat profiling using reference databases

• Test for mycoplasma contamination to ensure experimental reliability

• Develop conditional knockout models for temporal control of MEMO1 depletion

For in vivo models, consider tissue-specific conditional knockout approaches using Cre-lox systems targeted to specific cell types of interest. This comprehensive validation approach ensures that observed phenotypes are specifically attributable to MEMO1 modulation rather than off-target effects or compensatory mechanisms .

What analytical techniques are most effective for studying MEMO1 genetic interactions?

Studying MEMO1 genetic interactions requires sophisticated analytical approaches:

• Analysis of genome-wide CRISPR/Cas9 screening datasets from large cell line panels (>1000 cancer cell lines) stratified by MEMO1 expression levels

• Identification of genes showing differential essentiality in MEMO1-high versus MEMO1-low cell lines

• Statistical analysis using appropriate cutoffs (p-value <0.05) for differences in median essentiality scores

• Gene set enrichment analysis (GSEA) to identify pathways and processes enriched among MEMO1 genetic interactions

• Experimental validation of predicted interactions through targeted knockdowns in cell lines with varying MEMO1 expression

• Proliferation assays with real-time cell imaging systems (e.g., Incucyte S3) to quantify growth rates

• Data fitting using appropriate mathematical models (e.g., Logistic Growth equation)

This approach has successfully identified 18 iron-related genes with significant genetic interactions with MEMO1, including TFR2, SLC25A28, and ACO1, suggesting that high-MEMO1 cells are particularly vulnerable to disruptions in iron distribution and metabolism .

How can researchers effectively study the impact of MEMO1 on mitochondrial function and morphology?

To comprehensively assess MEMO1's impact on mitochondrial biology, researchers should implement:

• High-resolution confocal microscopy using mitochondrial markers (GRP75/HSPA9) and membrane potential-sensitive dyes (MitoTracker CM-H2Ros)

• Live-cell imaging to track mitochondrial dynamics and morphological changes in real-time

• Electron microscopy for ultrastructural analysis of mitochondrial morphology

• Mitochondrial fractionation followed by western blotting to assess mitochondrial protein composition

• Measurement of mitochondrial iron content using ICP-MS or mitochondria-targeted iron sensors

• Respirometry to assess mitochondrial respiratory capacity and efficiency

• Analysis under both basal conditions and stress conditions (e.g., iron chelation with 1μM DFX)

This multifaceted approach revealed that MEMO1 knockout cells display normal mitochondrial morphology under basal conditions but exhibit perinuclear mitochondrial clustering when treated with iron chelators, indicating MEMO1's specific role in maintaining mitochondrial function during iron depletion .

How does MEMO1 interact with the ERBB2 signaling pathway in cancer progression?

Investigating MEMO1-ERBB2 signaling interactions requires multiple methodological approaches:

• Co-immunoprecipitation studies to confirm direct interaction between MEMO1 and ERBB2

• Phosphoproteomic analysis to map signaling changes downstream of ERBB2 in MEMO1-modulated cells

• Live-cell imaging of cytoskeletal dynamics following ERBB2 activation with and without MEMO1

• Migration and invasion assays in response to ERBB2-activating ligands

• Analysis of lamellipodia formation and focal adhesion dynamics

• Assessment of microtubule stability at cell cortex using specialized imaging techniques

• Investigation of the MEMO1-RHOA-DIAPH1 signaling axis through activity probes and interaction studies

This research has established that MEMO1 relays activation signals from ERBB receptor heterodimers to the microtubule cytoskeleton, inducing lamellipodia growth and enabling cancer cell migration—a function that gave MEMO1 its name (mediator of ERBB2-driven cell motility 1) .

What methodologies can reveal the interactome of MEMO1 in different cellular contexts?

Researchers seeking to map the MEMO1 interactome should employ:

• Proximity-based labeling techniques (BioID, APEX) to identify proximal proteins in living cells

• Affinity purification coupled with mass spectrometry (AP-MS) using endogenous or tagged MEMO1

• Cross-linking mass spectrometry (XL-MS) to capture transient interactions

• Co-immunoprecipitation followed by western blotting for validation of specific interactions

• Yeast two-hybrid screening for direct binary interactions

• Fluorescence resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC) for in vivo interaction validation

• Comparative interactome analysis across different cell types, differentiation states, and disease models

These approaches should be applied in both normal and stress conditions, such as hypoxia, oxidative stress, or iron depletion, to reveal context-dependent interactions. Current research has identified interactions between MEMO1 and several iron-related proteins, including transferrin receptor 2 (TFR2), iron transporter mitoferrin-2 (SLC25A28), and iron response protein (ACO1) , as well as the copper chaperone Atox1 .

How might MEMO1 serve as a biomarker for cancer prognosis and treatment response?

Developing MEMO1 as a clinically useful biomarker requires rigorous methodological approaches:

• Retrospective analysis of patient tumor samples correlating MEMO1 expression with survival outcomes using appropriate statistical methods for survival analysis

• Multivariate analysis to determine if MEMO1 provides independent prognostic information beyond established markers

• Development of standardized immunohistochemistry protocols with validated scoring systems

• Assessment of MEMO1 expression in circulating tumor cells or cell-free DNA as liquid biopsy approaches

• Correlation of MEMO1 levels with response to specific therapeutic regimens, particularly those targeting iron metabolism

• Evaluation of MEMO1 in combination with other biomarkers to develop prognostic signatures

• Prospective clinical studies to validate the utility of MEMO1 as a biomarker

Clinical data have already shown strong correlation between increased MEMO1 expression and reduced patient survival in breast cancer . MEMO1 may serve as a biomarker of tumors particularly sensitive to therapies targeting iron metabolism, as MEMO1 overexpression appears to help maintain normal metabolism in cancer cells by increasing mitochondrial iron levels under hypoxic conditions .

What therapeutic strategies could target MEMO1-dependent vulnerabilities in cancer?

Developing targeted therapeutics against MEMO1-dependent cancer vulnerabilities requires:

• Identification of synthetic lethal interactions with MEMO1 through genome-wide screening approaches

• Focus on iron-related gene dependencies in MEMO1-high cancer cells (TFR2, SLC25A28, ACO1)

• Development of small molecule inhibitors of MEMO1-protein interactions or MEMO1 metal binding

• Testing of iron chelators specifically in MEMO1-overexpressing tumors

• Combination approaches targeting both MEMO1 and ERBB2 signaling pathways

• In vivo validation using patient-derived xenograft models with varying MEMO1 expression levels

• Assessment of both tumor growth inhibition and metastasis prevention

The specific genetic interactions of MEMO1 with iron-related genes suggest potential therapeutic windows where MEMO1-high cancer cells may be selectively vulnerable to disruptions in iron metabolism, offering promising avenues for targeted therapy development .