In previous studies, actin filaments in chemically-induced projections from human HAP1 cells and Drosophila S2 cells were shown to occupy luminal spaces of microtubules. These filaments display unique morphologies with shorter crossover spacings compared to canonical F-actin, pointing to the involvement of actin-binding proteins like cofilin.

In their study, the team at Bristol characterized lumenal F-actin in HAP1 cell projections and identified distinct filament types. Class I filaments, characterized by short crossover spacings and a smooth appearance, are indicative of cofilin binding. Class II filaments showed an F-actin-like power spectrum with a prominent meridional layer line, suggesting deviations from the standard F-actin helical morphology. A novel filament type, Class III, was also discovered which did not exhibit an F-actin-like power spectrum. Helical reconstructions and model docking confirmed that Class I filaments are indeed cofilin-bound F-actin, with Cofilin 1 (CFN1) being a likely major form in these cells.

To extend these findings to physiological conditions, the researchers investigated lumenal actin filaments in native human platelets. Platelets, which undergo significant mechanical stress during activation and biogenesis, possess a dynamic microtubule structure known as the marginal band. Using cryoET and cryoFIB-SEM, they observed bundled microtubule structures consistent with the marginal band and identified actin filaments in a subset of microtubules. Most of these filaments displayed the Class I morphology, confirming the presence of cofilin-bound F-actin. They also found less frequent examples of canonical cofilin-free actin filaments.

Interestingly, the study found no clear correlation between the presence of lumenal filaments and the number of protofilaments in microtubules, which was mostly 13, with some having 14. The absence of Class II and Class III filaments in platelets suggests that these may be more specific to the HAP1 cell system or require particular conditions for formation. This highlights the importance of studying the intermediate states of actin filaments and emphasizes the need for further structural studies of Class II and Class III filaments.

Overall, this research not only advances our understanding of cytoskeletal interactions but also sets the stage for future investigations into the role of lumenal F-actin in various cell types. Future studies will focus on exploring the impact of genetic mutations related to hereditary thrombocytopenia, particularly those affecting the cofilin pathway, and understanding the broader implications of lumenal actin in cellular mechanics. As high-resolution imaging techniques continue to evolve, they will undoubtedly play a crucial role in unraveling the complexities of filament dynamics within confined cellular environments.