Mechanics of Interpenetrating Biopolymer Networks in the Cytoskeleton and Biomolecular Condensates

Abstract

The cytoskeleton in eukaryotic cells is a dynamic network that provides the cells with structural support, the ability to resist deformation, and the locomotion of both cells themselves and the organelles within the cells. The cytoskeletal networks are primarily comprised of three types of cytoskeletal filaments, actin filaments, microtubules, and intermediate filaments; each of the three types plays distinct roles, and their interplay is responsible for maintaining normal cellular functions. Moreover, the cells also develop membrane-bound and membrane-less organelles to coordinate complex biochemical reactions in the cells. Compared with the regular organelles surrounded by lipid bilayers, the membrane-less organelles, also known as biomolecular condensates, are more adaptive and responsive to quick environmental changes. The dynamics and the formation mechanism of the condensates have drawn great interest over the past decade. Understanding the mechanics of both cytoskeletal networks and biomolecular condensates and their interplay is essential to learning normal cellular functions and provides important insights into the underlying mechanisms in pathologies of many diseases. In this thesis, we study the rheological properties of cytoskeletal networks and biomolecular condensates using microrheology. Using a variety of imaging-based techniques and analysis, we reveal the interactions between cytoskeleton and condensates and the interactions among different types of cytoskeletal filaments. Specifically, we begin our studies by reconstituting an interpenetrating cytoskeletal network composed of all three types of cytoskeletal filaments. We characterize the rheological properties of the network using microrheology. We find that the addition of vimentin intermediate filaments into the multi-component cytoskeletal network significantly prolongs the network relaxation time and transforms the network to be more solid-like; however, the addition of vimentin does not increase the network elastic modulus very much, which is dominated by actin filaments. This suggests a weak interaction between vimentin and actin filaments. Furthermore, we study the interactions between F-actin and vimentin in cells. Consistent with the findings from the in vitro study, we reveal the interactions between the two types of cytoskeletal filaments in their structures and find the interactions affect cell contractility. Lastly, we investigate the mechanics of biomolecular condensates and the interactions between the condensates and the cytoskeletal networks. From the microrheology measurements, we find FUS condensates become more gel-like while they are aging; the transition is potentially associated with the pathology of neurodegenerative diseases. Moreover, we find that exposure to short-wavelength light can accelerate gelation. To study the interactions between cytoskeleton and condensates, we induce stress granules in cells by chemical stimuli and observe the formation and dynamics of the stress granules affected by microtubules. We demonstrate the dramatic difference in the condensate formation and dynamics when microtubules are intact or disassembled. These results alter our understanding of the contributions of the cytoskeletal filaments and their interplay with condensates and help rationalize the complex behaviors of these components in cells.