Proteomic Profiling of Skin Lamellar Bodies During Keratinocyte Differentiation

1. Overview: This project aims to characterize skin lamellar bodies (LBs)—specialized lysosome-related organelles crucial for skin barrier formation—during keratinocyte differentiation. We will use Mass Spectrometry-based Proteomics to uncover the molecular composition and biogenesis of LBs, leveraging recent advances in protein profiling. Funding, facilities, and ethics approvals will be confirmed prior to study initiation. The estimated timeline is 6 months, with a commitment to open data sharing upon completion.

2. Introduction: The skin is the body’s largest organ and is made up of several distinct layers[1]. The topmost layer, the epidermis, serves as the primary shield against the environment, while the deeper layer, the dermis, provides structural support and houses essential tissues. Together, these layers form a complex barrier that is vital for life on land, helping defend against harmful substances, prevent infections, and maintain proper hydration by reducing water loss [1].

Central to the formation and maintenance of this barrier are skin lamellar bodies (LBs), which are specialized lysosome-related organelles (LROs) unique to keratinocytes[2]. LBs play an essential role in delivering lipids, enzymes, and antimicrobial peptides to the stratum corneum, thereby facilitating the assembly of the extracellular lipid matrix that underpins barrier function. The stratum corneum is the outermost layer of the epidermis[3].

LBs are generated during the terminal differentiation of keratinocytes, a process marked by profound cellular remodeling, including cytoskeletal reorganization, organelle dissolution, and extensive plasma membrane modifications [4-6]. The biogenesis of LBs is tightly regulated by differentiation cues, such as calcium influx, and characterized by the selective packaging of cargo distinct from that of conventional lysosomes and other LROs[7]. Proteomics has revealed that LB formation coincides with the upregulation of genes involved in lipid metabolism, membrane trafficking, and vesicular transport, suggesting a complex regulatory network governing their assembly and function[2].

Despite their critical role in skin physiology, the molecular mechanisms underlying LB biogenesis, cargo selection, and secretion remain incompletely understood. Dysregulation of LB formation or function has been implicated in a spectrum of dermatological disorders, including atopic dermatitis, Netherton syndrome, and ichthyoses, underscoring the clinical relevance of elucidating LB biology[8]. Notably, mutations in genes encoding LB-associated proteins can disrupt barrier integrity, leading to increased transepidermal water loss and heightened susceptibility to infection and inflammation.

This project seeks to systematically characterize the proteomic landscapes of LBs during keratinocyte differentiation. By integrating with high-resolution mass spectrometry, we aim to identify key molecular determinants of LB biogenesis and function, delineate the temporal dynamics of their assembly, and uncover novel regulators of skin barrier formation. The insights gained will not only advance our fundamental understanding of epidermal biology but may also inform the development of targeted therapies for skin barrier disorders.

2.1. Research Questions:

What are the proteomic profiles of LBs during keratinocyte differentiation?
Which proteins are key regulators of LB biogenesis and function?

2.2. Hypotheses:

H1: Keratinocyte differentiation induces unique proteomic signatures in lamellar bodies.
H2: Specific enzymes, membrane proteins, and lipids are upregulated during lamellar body formation.

2.3. Impact Statement:
Deepening our understanding of lamella body biogenesis has the potential to transform our knowledge of skin barrier physiology and to unravel the molecular basis of skin diseases, paving the way for innovative diagnostic and therapeutic strategies, regardless of the experimental outcomes.

3. Methods

3.1. Participants/Sample

Generation of 3D human equivalent skin organoids. A total of three to four organoids will be collected at the time points listed below. Since this is a pilot study, no power analysis will be performed at this stage, as it requires a sample size of 6 or more, which is beyond the scope of the proposal.

3.2. Materials and Procedures

Generation and Harvesting of Organoids: Primary neonatal human keratinocytes and immortalized keratinocyte cell lines will be used to generate three-dimensional human skin organoids in vitro. 3D organoids will be harvested at different stages of the keratinocyte differentiation (day 6, day 9, day 12). The organoid will be divided into three parts. One part will be used for Immunohistochemistry experiments, the second for Transmission Electron Microscopy, and the third for the isolation of lamellar bodies.
Isolation of lamellar bodies: The organoids will be gently dissociated using enzymatic digestion, specifically with Accutase. Following this process, the cells will be homogenized in a sucrose buffer for subcellular fractionation. Lamellar bodies will be isolated from the cells using differential centrifugation. All procedures will be carried out at cold temperatures to prevent degradation of subcellular components. Purified lamellar bodies will be assessed by Western blotting.

Immunohistochemistry: for visualizing the differentiation stage.
Transmission Electron Microscopy for LB visualization.
Proteomics (mass spectrometry) to identify protein cargo.
Data collection in a controlled laboratory environment.

3.3. Planned Analyses

Analysis pipeline and pre-processing steps are as follows: 1. Sample Preparation: Harvest organoids at differentiation stages (day 6, day 9, day 12) and dissociate using Accutase. 2. Subcellular Fractionation - Homogenize cells in a sucrose buffer and use differential centrifugation to isolate lamellar bodies. 3. Immunohistochemistry - Fix and stain organoid sections to visualize differentiation markers using microscopy. 4. Transmission Electron Microscopy (TEM) - Prepare and section samples of lamellar bodies for high-resolution imaging. TEM will be performed by our collaborators. 5. Proteomics Analysis- Isolate proteins from lamellar bodies, prepare samples, and analyze using mass spectrometry. Samples will be sent out for proteomics 6. Data Integration and Analysis- Compile proteomics data, perform bioinformatics analysis, and integrate findings. 7. Result Validation - Validate key findings with additional assays and conduct statistical analysis. 8. Reporting and Visualization- Visualize data and compile a report summarizing results related to keratinocyte differentiation and lamellar body characteristics.
Statistical tests: A two-way ANOVA will be performed.
IF-THEN: If novel LB-associated proteins are found, validate by knockdown/overexpression.
Hypothesis-specific: Compare undifferentiated vs. differentiated keratinocytes for LB profiles.

3.4. Ethics and Data Management

No ethical measurements were needed as this study does not involve human samples.
Confidentiality maintained; upon manuscript publication, data will be made available to the public.

4. Budget

Materials: Cell culture and generation of 3D organoids: $1500
Isolation Lamellar Bodies: $1000
Proteomics: $2500

5. References

1. Abdallah, F., L. Mijouin, and C. Pichon, Skin Immune Landscape: Inside and Outside the Organism. Mediators Inflamm, 2017. 2017: p. 5095293.

2. Mahanty, S. and S.R.G. Setty, Epidermal Lamellar Body Biogenesis: Insight Into the Roles of Golgi and Lysosomes. Front Cell Dev Biol, 2021. 9: p. 701950.

3. Feingold, K.R., Lamellar bodies: the key to cutaneous barrier function. J Invest Dermatol, 2012. 132(8): p. 1951-3.

4. Akiyama, M., The roles of ABCA12 in keratinocyte differentiation and lipid barrier formation in the epidermis. Dermatoendocrinol, 2011. 3(2): p. 107-12.

5. Akinduro, O., et al., Constitutive Autophagy and Nucleophagy during Epidermal Differentiation. J Invest Dermatol, 2016. 136(7): p. 1460-1470.

6. Akiyama, M., The roles of ABCA12 in epidermal lipid barrier formation and keratinocyte differentiation. Biochim Biophys Acta, 2014. 1841(3): p. 435-40.

7. Mahanty, S., et al., Keratinocyte differentiation promotes ER stress-dependent lysosome biogenesis. Cell Death Dis, 2019. 10(4): p. 269.

8. Mahanty, S., Skin lamellar bodies: a unique set of lysosome-related organelles. Front Cell Dev Biol, 2025. 13: p. 1597696.

9. The cover image is created with new AI-beta BioRender.com

Proteomic Profiling of Skin Lamellar Bodies During Keratinocyte Differentiation

Proposal

Proposal

Proposal

Proposal

Proteomic Profiling of Skin Lamellar Bodies During Keratinocyte Differentiation

Proposal

Proposal

Funding Progress

Nonprofit Recipient

Topics

DOI

Funding Progress

Nonprofit Recipient

Topics

DOI