The Human Lens Fiber Cytoskeletome Reveals Neuronal Signatures and the Presence of Chaperonins, Proteasome, Signaling, and Redox Regulators

Purpose: Morphogenesis, growth, shape, mechanics, and transparency of the ocular lens critically depend on cytoskeletal networks and cell adhesive interactions. However, the molecular composition of these cellular components of lens fibers remains to be defined in depth. This study is aimed at providing an unbiased and comprehensive characterization of cytoskeletal and cytoskeleton-associated proteins in human lens fibers. Methods: Transparent lenses from young adult human donors (ages 21 and 31), with capsules removed, were processed to enrich the cytoskeletome fraction, which was then trypsin-digested and subjected to proteomics analysis using a Q Exactive HF Orbitrap LC-MS/MS spectrometer. Selected proteins were validated by immunoblotting and immunofluorescence. Gene Ontology enrichment analysis was performed to classify the identified proteins. Results: Proteomic analysis identified 530 proteins in the human lens fiber cytoskeletome, including known and lesser-known cytoskeletal, membrane-associated, adhesive, scaffolding, signaling, and metabolic proteins. Notably, several neuron preferred proteins, chaperonins, and proteasome, signaling, and redox regulators were enriched in the cytoskeletal interactome. Many of the neuron preferred proteins were localized to the cortical lens fiber cells. In addition, certain cytoskeletal protein profiles differed between human and mouse lenses. Conclusions: This study provides the first comprehensive profile of the human lens fiber cytoskeletome. It highlights the presence of neuron-enriched cytoskeletal, adhesive, and scaffolding proteins; cytoskeletal chaperonins; proteasome, signaling, and redox regulators; and both canonical and lesser-known cytoskeletal proteins. These findings suggest that a diverse, complex, and dynamically regulated cytoskeletal network contributes to lens fiber cell architecture, adhesion, trafficking, mechanics, and clarity.