Cellular proteins do not work in isolation. Instead, they often function as part of large macromolecular complexes, which are transported and concentrated into specific cellular compartments and function in a highly crowded environment. A central theme of modern cell biology is to understand how such macromolecular complexes are assembled efficiently and find their destinations faithfully. In this Opinion article, we will focus on HEAT repeats, flexible arrays of amphiphilic helices found in many eukaryotic proteins, such as karyopherins and condensins, and discuss how these uniquely designed helical repeats might underlie dynamic protein–protein interactions and support cellular functions in crowded environments. We will make bold speculations on functional similarities between the action of HEAT repeats and intrinsically disordered regions (IDRs) in macromolecular phase separation. Potential contributions of HEAT–HEAT interactions, as well as cooperation between HEATs and IDRs, to mesoscale organelle assembly will be discussed.

Dynamic morphological changes of intracellular organelles are often regulated by protein phosphorylation or dephosphorylation^1–6. Phosphorylation modulates stereospecific interactions among structured proteins, but how it controls molecular interactions among unstructured proteins and regulates their macroscopic behaviours remains unknown. Here we determined the cell cycle-specific behaviour of Ki-67, which localizes to the nucleoli during interphase and relocates to the chromosome periphery during mitosis. Mitotic hyperphosphorylation of disordered repeat domains of Ki-67 generates alternating charge blocks in these domains and increases their propensity for liquid–liquid phase separation (LLPS). A phosphomimetic sequence and the sequences with enhanced charge blockiness underwent strong LLPS in vitro and induced chromosome periphery formation in vivo. Conversely, mitotic hyperphosphorylation of NPM1 diminished a charge block and suppressed LLPS, resulting in nucleolar dissolution. Cell cycle-specific phase separation can be modulated via phosphorylation by enhancing or reducing the charge blockiness of disordered regions, rather than by attaching phosphate groups to specific sites.

Clathrin-mediated endocytosis (CME) proceeds through a series of morphological changes of the plasma membrane induced by a number of protein components. Although the spatiotemporal assembly of these proteins has been elucidated by fluorescence-based techniques, the protein-induced morphological changes of the plasma membrane have not been fully clarified in living cells. Here, we visualize membrane morphology together with protein localizations during CME by utilizing high-speed atomic force microscopy (HS-AFM) combined with a confocal laser scanning unit. The plasma membrane starts to invaginate approximately 30 s after clathrin starts to assemble, and the aperture diameter increases as clathrin accumulates. Actin rapidly accumulates around the pit and induces a small membrane swelling, which, within 30 s, rapidly covers the pit irreversibly. Inhibition of actin turnover abolishes the swelling and induces a reversible open–close motion of the pit, indicating that actin dynamics are necessary for efficient and irreversible pit closure at the end of CME. Cells communicate with their environments via the plasma membrane and various membrane proteins. Clathrin-mediated endocytosis (CME) plays a central role in such communication and proceeds with a series of multiprotein assembly, deformation of the plasma membrane, and production of a membrane vesicle that delivers extracellular signaling molecules into the cytoplasm. In this study, we utilized our home-built correlative imaging system comprising high-speed atomic force microscopy (HS-AFM) and confocal fluorescence microscopy to simultaneously image morphological changes of the plasma membrane and protein localization during CME in a living cell. The results revealed a tight correlation between the size of the pit and the amount of clathrin assembled. Actin dynamics play multiple roles in the assembly, maturation, and closing phases of the process, and affects membrane morphology, suggesting a close relationship between endocytosis and dynamic events at the cell cortex. Knock down of dynamin also affected the closing motion of the pit and showed functional correlation with actin.

Importin-β mediates protein transport across the nuclear envelope through the nuclear pore complex (NPC) by interacting with components of the NPC, called nucleoporins, and a small G protein, Ran. Although there is accumulated knowledge on the specific interaction between importin-β and the Phe-Gly (FG) motif in the nucleoporins as well as the effect of RanGTP on this interaction, the molecular mechanism by which importin-β shuttles across the nuclear envelope through the NPC is unknown. In this study, we focused on four binding pockets of importin-β for the FG motifs and characterized the interaction using a single-molecule force-measurement technique with atomic-force microscopy. The results from a series of importin-β mutants containing amino acid substitutions within the FG-binding pockets demonstrate that the individual FG-binding pockets have different affinities to FG-Nups (Nup62 and Nup153) and different sensitivities to RanGTP; the binding of RanGTP to the amino-terminal domain of importin-β induces the conformational change of the entire molecule and reduces the affinity of some of the pockets but not others. These heterogeneous characteristics of the multiple FG-binding pockets may play an important role in the behavior of importin-β within the NPC. Single-molecule force measurement using the entire molecule of an NPC from a Xenopus oocyte also implies that the reduction of the affinity by RanGTP really occurs at the nucleoplasmic side of the entire NPC.

Together with lamellipodia and stress fibers, a dynamic network of actin filaments in the cell cortex plays a major role in the maintenance of cell morphology and motility. In contrast to lamellipodia, which have been well studied in various motile cells, the dynamics of actin filaments in the cell cortex have not yet been clarified due to a lack of proper imaging techniques. Here, we utilized high-speed atomic force microscopy for live-cell imaging and analyzed cortical actin dynamics in living cells. We successfully measured the polymerization rate and the frequency of filament synthesis in living COS-7 cells, and examined the associated effects of various inhibitors and actin-binding proteins. Actin filaments are synthesized beneath the plasma membrane and eventually descend into the cytoplasm. The inhibitors, cytochalasin B inhibited the polymerization, while jasplakinolide, inhibited the turnover of actin filaments as well as descension of the newly synthesized filaments, suggesting that actin polymerization near the membrane drives turnover of the cortical actin meshwork. We also determined how actin turnover is maintained and regulated by the free G-actin pool and G-actin binding proteins such as profilin and thymosin β4, and found that only a small amount of free G-actin was present in the cortex. Finally, we analyzed several different cell types, and found that the mesh size and the orientation of actin filaments were highly divergent, indicating the involvement of various actin-binding proteins in the maintenance and regulation of cortical actin architecture in each cell type.