MARCKSL1 Human

What is MARCKSL1 and how does it differ from MARCKS?

MARCKSL1 (MARCKS-like protein 1), also known as MLP, MRP, Brain Protein F52, or MacMARCKS, is a 20 kDa protein that shares strong homology and functionality with MARCKS (Myristoylated Alanine-Rich C-Kinase Substrate). Both proteins contain similar effector domains (ED) that bind F-actin, Ca²⁺/calmodulin, and acidic phospholipids, as well as N-terminal myristoylation consensus sequences .

The key differences include:

• MARCKSL1 has a lower alanine content than MARCKS, which may result in functional differences

• MARCKSL1 exhibits a distinct distribution pattern in the brain compared to MARCKS

• While both proteins are involved in cytoskeletal regulation, they may have tissue-specific functions and expression patterns

What are the primary molecular interactions of MARCKSL1?

MARCKSL1 engages in several critical molecular interactions depending on its phosphorylation state:

• Actin cytoskeleton interaction: When unphosphorylated, MARCKSL1 can bundle and stabilize F-actin, increasing filopodium dynamics

• Membrane association: Similar to MARCKS, MARCKSL1 can associate with the plasma membrane through its effector domain and myristoylation

• Ca²⁺/calmodulin binding: The effector domain of MARCKSL1 binds Ca²⁺/calmodulin, which regulates its association with the membrane and actin cytoskeleton

• Phosphorylation-dependent interactions: Upon phosphorylation, MARCKSL1 changes its binding characteristics, altering its interactions with cytoskeletal elements and membrane components

These interactions collectively contribute to MARCKSL1's roles in cellular processes such as migration, morphology, and proliferation.

What antibodies and detection methods are most effective for studying MARCKSL1?

For optimal detection of MARCKSL1 in experimental settings, researchers can utilize specifically developed antibodies such as MARCKSL1 (D4I9P) Rabbit mAb. Based on technical data from established protocols:

Recommended Antibody Characteristics:

• Recombinant antibodies offer superior lot-to-lot consistency and continuous supply

• For Western blotting applications, a 1:1000 dilution is typically effective

• Expected molecular weight for detection is approximately 40 kDa

Application-Specific Guidance:

Species Cross-Reactivity:

• Human (H)

• Mouse (M)

When designing experiments requiring MARCKSL1 protein detection, researchers should consider the specificity and sensitivity of the selected antibody and validate detection in their specific experimental system.

What viral constructs are recommended for MARCKSL1 overexpression or knockdown studies?

Based on successful protocols in published research, the following viral constructs have proven effective for manipulating MARCKSL1 expression in experimental systems:

For MARCKSL1 Knockdown:

• shRNA constructs targeting MARCKSL1 (TRC# 0000157260, 0000156469, 0000152478) have been successfully employed in combination with rtTA and Ngn2-t2a-Blasticidin Resistance (driven by Tet-on promoter) systems

• Empty pLKO.1 vector should be used as an infection control

For MARCKSL1 Overexpression:

• Lentivirus encoding human cDNA of wild-type MARCKSL1 cloned under a Tet-on promoter and followed by IRES-driven EGFP has been effectively implemented

• This construct is typically co-infected with rtTA and Ngn2-t2a-Puromycin Resistance-expressing virus

• Control lentivirus containing only IRES-EGFP should be used as experimental control

These constructs allow for inducible expression systems that provide temporal control over MARCKSL1 manipulation, facilitating more precise experimental designs.

How does MARCKSL1 influence dendritic morphology and synapse formation in neurons?

MARCKSL1 plays a crucial role in regulating neuronal dendritic morphology and synapse formation, particularly evident in experiments investigating valproic acid (VPA)-induced defects in neuronal development:

Mechanisms of Action:

• MARCKSL1 overexpression has been shown to completely reverse VPA-mediated defects in dendritic morphology and synapse numbers in human neurons derived from pluripotent stem cells

• While MARCKSL1 expression had no measurable effect in control cells, it fully rescued VPA-mediated reduction in membrane capacitance (Cm) without affecting membrane resistance (Rm), membrane potential (Vm), or action potential firing

Synaptic Function Effects:

• MARCKSL1 overexpression completely reversed VPA-induced reductions in:

  • Surface levels of synaptic receptors (measured by AMPAR responses to puff-applied AMPA)

  • Synaptic strength (measured by AMPAR EPSC amplitude)

These findings suggest that MARCKSL1's cytoskeletal regulatory functions are essential for proper dendritic development and synaptogenesis, consistent with its role in modulating actin dynamics. Notably, MARCKSL1 overexpression did not reverse VPA-induced increases in synapse size, indicating pathway specificity in its rescue effects .

What experimental models are most suitable for studying MARCKSL1 function in neural development?

Several experimental models have proven valuable for investigating MARCKSL1 function in neural development:

Human Pluripotent Stem Cell-Derived Neurons:

• Particularly effective for studying human-specific aspects of MARCKSL1 function

• Rapid conversion into functional neurons can be achieved through ectopic expression of proneural transcription factors like Ngn2

• These models allow for genetic manipulation (overexpression/knockdown) and pharmacological intervention studies

Developing Retina Models:

• Useful for studying MARCKSL1's role in cell proliferation during development

• Research has shown that MARCKSL1 expression decreases as retinal development progresses

Experimental Design Considerations:

• Blinded experimental design is critical to prevent bias in analyzing MARCKSL1-related phenotypes

• Appropriate controls must include both genetic manipulation controls (empty vectors) and treatment controls

• Time-point selection is crucial as MARCKSL1's effects can vary with developmental stage (e.g., overexpression at day 19 with treatment at day 21 showed more profound rescue effects than simultaneous intervention)

When designing studies to investigate MARCKSL1 function in neural development, researchers should consider the specific temporal and spatial contexts relevant to their research questions.

What is the role of MARCKSL1 in cancer progression and how can it be studied?

MARCKSL1 has emerged as a significant factor in cancer biology, with multiple lines of evidence suggesting roles in cancer progression:

Oncological Significance:

• MARCKSL1 may serve as a prognostic marker in lymph node-negative breast cancer

• It regulates migration and actin dynamics in prostate cancer cells in response to phosphorylation by JNK

Experimental Approaches for Cancer Studies:

• Expression Analysis in Patient Samples:

  • Immunohistochemistry and tissue microarray approaches to correlate MARCKSL1 expression with clinical outcomes

  • RNA sequencing to identify expression patterns across cancer subtypes

• Functional Studies in Cancer Cell Lines:

  • Migration and invasion assays following MARCKSL1 knockdown or overexpression

  • Cytoskeletal visualization techniques to assess changes in cell morphology and actin dynamics

  • Phosphoproteomic analysis to understand the activation of MARCKSL1 in cancer contexts

• Animal Models:

  • Xenograft models with MARCKSL1-modulated cancer cells

  • Transgenic animal models with tissue-specific MARCKSL1 alterations

When designing cancer-related MARCKSL1 studies, researchers should consider integrating multiple experimental approaches to establish both correlation and causation in the relationship between MARCKSL1 and cancer progression.

How does MARCKSL1 contribute to VPA-induced developmental neurotoxicity?

Valproic acid (VPA), an anticonvulsant and mood stabilizer, can cause developmental neurotoxicity. Research has revealed that MARCKSL1 is a key mediator in this process:

Molecular Mechanisms:

• VPA treatment downregulates MARCKSL1 expression through HDAC inhibition, leading to cytoskeletal dysregulation

• This dysregulation results in impaired dendritic morphology, reduced synapse numbers, and compromised synaptic function

Experimental Evidence:

• Neurons treated with VPA show reduced dendritic arborization and synapse formation

• These VPA-induced pathologies can be completely reversed by MARCKSL1 overexpression, confirming MARCKSL1's central role in the pathogenic mechanism

• MARCKSL1 rescue effects include normalization of:

  • Dendritic arborization

  • Synapse numbers

  • Membrane capacitance (Cm)

  • AMPAR responses to puff-applied AMPA

  • AMPAR EPSC amplitude

Methodological Approach for Study:

• Human pluripotent stem cell-derived neurons provide an excellent model system

• Time-course experiments are crucial to distinguish between direct and secondary effects

• Combined electrophysiological and morphological analyses provide comprehensive assessment

These findings suggest that MARCKSL1 restoration could represent a potential therapeutic strategy for VPA-induced neurodevelopmental disorders, highlighting the importance of cytoskeletal regulation in neural development.

How do phosphorylation states regulate MARCKSL1 function in different cellular contexts?

MARCKSL1's functional versatility largely depends on its phosphorylation state, which acts as a molecular switch controlling its interactions and cellular functions:

Phosphorylation-Dependent Mechanisms:

• When unphosphorylated, MARCKSL1's effector domain can bind and cross-link F-actin, promoting actin polymerization and stabilization

• Upon phosphorylation, MARCKSL1 can dissociate from the membrane, altering its interactions with the cytoskeleton

• In response to phosphorylation by JNK, MARCKSL1 regulates migration and actin dynamics in neuronal and prostate cancer cells

Context-Specific Functions:

• In filopodium dynamics, MARCKSL1 bundles and stabilizes F-actin upon phosphorylation, enhancing filopodial activity

• In developing neurons, the phosphorylation state of MARCKSL1 affects its ability to regulate dendritic arborization and synaptogenesis

• During cell migration, cycles of phosphorylation and dephosphorylation likely coordinate MARCKSL1's contribution to cytoskeletal remodeling

To effectively study these phosphorylation-dependent functions, researchers should employ phosphorylation-specific antibodies, phosphomimetic and phosphodeficient mutants, and targeted kinase inhibitors in their experimental designs.

What are the potential nuclear functions of MARCKSL1 and how can they be investigated?

While MARCKSL1 is primarily recognized for its cytoplasmic and membrane-associated roles, emerging evidence suggests potential nuclear functions that remain largely unexplored:

Evidence for Nuclear Roles:

• The effector domain (ED) of MARCKSL1, similar to MARCKS, acts as a nuclear localization signal, suggesting possible nuclear functions

• This domain shares homology with a region in diacylglycerol kinase zeta (DGKζ) that binds to and modulates retinoblastoma protein (Rb) function

Potential Nuclear Functions:

• Modulation of gene expression through direct or indirect interaction with transcriptional machinery

• Regulation of nuclear PIP₂ localization and nuclear phosphoinositide signaling

• Possible involvement in cell cycle regulation through interaction with cell cycle proteins like Rb

Recommended Investigative Approaches:

• Subcellular Fractionation and Imaging:

  • Immunofluorescence with phosphorylation-specific antibodies

  • Live-cell imaging with fluorescently tagged MARCKSL1 constructs

  • Super-resolution microscopy to visualize nuclear localization patterns

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation to identify nuclear binding partners

  • Chromatin immunoprecipitation (ChIP) to assess potential DNA associations

  • Proximity ligation assays to visualize interactions in situ

• Functional Studies:

  • Gene expression analysis following nuclear-targeted MARCKSL1 manipulation

  • Cell cycle analysis in contexts of MARCKSL1 overexpression or knockdown

  • Nuclear phosphoinositide measurements in response to MARCKSL1 modulation

These nuclear functions of MARCKSL1 represent an exciting frontier in understanding this multifaceted protein beyond its established cytoskeletal regulatory roles.

How does MARCKSL1 contribute to tissue development and regenerative processes?

MARCKSL1 has emerged as a key player in various developmental and regenerative contexts, with evidence supporting its importance across diverse tissue types:

Developmental Roles:

• In the developing retina, MARCKSL1 regulates cell proliferation, with expression decreasing as retinal development progresses

• MARCKSL1 contributes to brain development through its cytoskeletal regulatory functions

• The protein has been implicated in kidney, blood vessel, and muscle development

Regenerative Functions:

• The axolotl homolog of MARCKSL1 (AxMLP) functions as a secreted factor in limb regeneration, suggesting novel modes of action beyond the traditional intracellular roles

• MARCKSL1 may be involved in multiple regenerative processes including nerve, auditory hair cell, fin/limb, and lung regeneration

Molecular Basis of Developmental and Regenerative Contributions:

• MARCKSL1's ability to regulate the actin cytoskeleton facilitates cell migration and morphological changes essential for tissue development

• Its role in cell proliferation may contribute to the generation of new cells during both development and regeneration

• Potential interactions with development-relevant signaling pathways, such as retinoic acid (RA) signaling

Understanding MARCKSL1's functions in development and regeneration could provide valuable insights for regenerative medicine applications, particularly in contexts such as spinal cord injury .

What regulatory pathways control MARCKSL1 expression and activity during development?

Several regulatory pathways modulate MARCKSL1 expression and activity throughout development, creating a complex network of controls:

Transcriptional Regulation:

• Developmental stage-specific expression patterns suggest temporal transcriptional control mechanisms

• In the developing retina, MARCKSL1 expression decreases as development progresses, indicating developmental timing-specific regulation

Epigenetic Regulation:

• Histone deacetylase (HDAC) inhibition by valproic acid (VPA) leads to downregulation of MARCKSL1, suggesting epigenetic control of its expression

• This VPA-mediated suppression contributes to developmental neurotoxicity, highlighting the importance of proper MARCKSL1 regulation

Post-translational Modifications:

• Phosphorylation by protein kinase C (PKC) and c-Jun N-terminal kinase (JNK) regulates MARCKSL1 activity

• These modifications affect MARCKSL1's interactions with the membrane, actin cytoskeleton, and potentially other binding partners

Signaling Pathway Interactions:

• Retinoic acid (RA) signaling affects MARCKSL1 function in rat hippocampal cells, causing translocation from the membrane to the cytosol

• RA plays significant roles in numerous regenerative processes, suggesting a potential mechanistic link between MARCKSL1 and regeneration

Investigating these regulatory pathways requires integrated approaches combining transcriptional, epigenetic, and post-translational analyses across developmental timepoints.

What novel methodologies could advance our understanding of MARCKSL1 function?

Emerging technologies and methodological approaches offer promising avenues to deepen our understanding of MARCKSL1's diverse functions:

Advanced Imaging Techniques:

• Super-resolution microscopy to visualize MARCKSL1-actin interactions at nanoscale resolution

• Live-cell imaging with optogenetic control of MARCKSL1 phosphorylation to study dynamic changes in real-time

• Expansion microscopy to better visualize subcellular localization and protein interactions

Genome Editing Approaches:

• CRISPR/Cas9-mediated generation of phosphorylation site-specific mutants in endogenous MARCKSL1

• Creation of conditional knockout models to study tissue-specific functions

• Knock-in of fluorescent tags at the endogenous locus to track native MARCKSL1 dynamics

Single-Cell Analysis:

• Single-cell transcriptomics to identify cell-specific expression patterns during development

• Single-cell proteomics to analyze MARCKSL1 interaction networks in specific cellular contexts

• Spatial transcriptomics to map MARCKSL1 expression patterns in developing tissues

Computational Approaches:

• Molecular dynamics simulations to understand conformational changes upon phosphorylation

• Machine learning algorithms to identify novel MARCKSL1 interaction partners from proteomics datasets

• Systems biology approaches to integrate MARCKSL1 into broader signaling networks

These methodological advances would help address current knowledge gaps regarding MARCKSL1's complex functions in different cellular contexts and developmental stages.

How should researchers design experiments to investigate contradictory findings about MARCKSL1 function?

The multifaceted nature of MARCKSL1 has led to apparently contradictory findings across different experimental systems. To address these contradictions effectively:

Experimental Design Principles:

• Contextual Considerations:

  • Cell type-specific effects should be explicitly addressed by using multiple cell types

  • Developmental timing should be carefully controlled and reported

  • Phosphorylation status should be monitored alongside functional outcomes

• Methodological Triangulation:

  • Employ multiple complementary techniques to study the same phenomenon

  • Combine in vitro, cellular, and in vivo approaches when possible

  • Use both loss-of-function and gain-of-function approaches

• Quantitative Analysis:

  • Report effect sizes and confidence intervals, not just statistical significance

  • Consider dose-response relationships for manipulations of MARCKSL1

  • Employ appropriate statistical methods for complex experimental designs

Specific Approaches for Common Contradictions:

• For contradictory findings on cytoskeletal effects:

  • Directly measure actin dynamics using techniques like FRAP or live F-actin probes

  • Consider the phosphorylation state and local Ca²⁺ levels when interpreting results

  • Examine both acute and chronic effects of MARCKSL1 manipulation

• For inconsistent developmental phenotypes:

  • Carefully document developmental stages using standardized criteria

  • Consider genetic background effects in animal models

  • Examine compensatory mechanisms that may mask phenotypes in different contexts

All experiments should follow blinded designs whenever possible, as demonstrated in successful studies of MARCKSL1 function in neuronal development .