Modulation of Microtubule Curvature under Different Cellular Conditions Revealed by In-Cell Ceryo-Electron Tomography

Abstract

With persistence lengths in the range of 1–6 mm, microtubules (MTs) are the most rigid polymer of all cytoskeletal elements. However, inside cells they often exhibit highly curved shapes. Thus, it is tempting to speculate that the curvature of MTs in vivo are modulated by cellular factors resulting in bending at micrometer length scales. In order to better characterize the curvature of MTs and its potential causes in vivo, we quantified the curvature of cellular MTs in their native state from frozen-hydrated HeLa cells in interphase and mitosis as model systems using cryo-electron tomography (cryo-ET). We show that cellular MTs are highly curved beyond the limit posed by the polymer's intrinsic material's property. In fact, the apparent persistence lengths measured from the curved MTs are ∼100-1000 times lower than the persistence length obtained from thermal fluctuations alone, indicating prevalence of high magnitude of nonthermal forces in vivo. By using specific inhibitors, we pinpoint the sources of these forces that predominantly include actin-mediated compressive forces, ATP-dependent motor activities and microtubule polymerization. MT curvature was also found to vary depending on the cellular state, with mitotic MTs being relatively less curved implying that the magnitude of nonthermal forces have different regimes across cellular states in vivo. Notably, in-cell cryo-ET provides a glimpse into the native organization of MTs intertwined within the composite network of other cytoskeletal filaments and reveals differences in their organization across different cell cycle stages and cell types. We demonstrate that the presence of a heterogeneous elastic actin matrix and its organization is one of the main factors that influence the MT curvature in vivo.