Maruša Lokar1,*, Doron Kabaso1,2,*, Nataša Resnik3, Kristina Sepcic5, Veronika Kralj-Iglic4,6, Peter Veranic3, Robert Zorec2, Aleš Iglic1,6 1Laboratory of Biophysics, Faculty of Electrical Engineering, 2Laboratory of Neuroendocrinology-Molecular Cell Physiology, Faculty of Medicine, 3Institute of Cell Biology, Faculty of Medicine, 4Faculty of Health Sciences, 5Department of Biology, Biotechnical Faculty, 6Laboratory of Clinical Biophysics, Department of Orthopedic Surgery, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia*These authors equally share first authorshipAbstract: Intercellular membrane nanotubes (ICNs) are highly curved tubular structures that connect neighboring cells. The stability of these structures depends on the inner cytoskeleton and the cell membrane composition. Yet, due to the difficulty in the extraction of ICNs, the cell membrane composition remains elusive. In the present study, a raft marker, ostreolysin, revealed the enrichment of cholesterol-sphingomyelin membrane nanodomains along ICNs in a T24 (malignant) urothelial cancer cell line. Cholesterol depletion, due to the addition of methyl-β-cyclodextrin, caused the dispersion of cholesterol-sphingomyelin membrane nanodomains and the retraction of ICNs. The depletion of cholesterol also led to cytoskeleton reorganization and to formation of actin stress fibers. Live cell imaging data revealed the possible functional coupling between the change from polygonal to spherical shape, cell separation, and the disconnection of ICNs. The ICN was modeled as an axisymmetric tubular structure, enabling us to investigate the effects of cholesterol content on the ICN curvature. The removal of cholesterol was predicted to reduce the positive spontaneous curvature of the remaining membrane components, increasing their curvature mismatch with the tube curvature. The mechanisms by which the increased curvature mismatch could contribute to the disconnection of ICNs are discussed.Keywords: cyclodextrins, T24 urothelial cancer cell line, intercellular membrane nanotubes, cholesterol-sphingomyelin membrane nanodomains