Fluorescence polarization microscopy images both the intensity and orientation of fluorescent dipoles and plays a vital role in studying molecular structures and dynamics of bio-complexes. However, current techniques remain difficult to resolve the dipole assemblies on subcellular structures and their dynamics in living cells at super-resolution level. Here we report polarized structured illumination microscopy (pSIM), which achieves super-resolution imaging of dipoles by interpreting the dipoles in spatio-angular hyperspace. We demonstrate the application of pSIM on a series of biological filamentous systems, such as cytoskeleton networks and λ-DNA, and report the dynamics of short actin sliding across a myosin-coated surface. Further, pSIM reveals the side-by-side organization of the actin ring structures in the membrane-associated periodic skeleton of hippocampal neurons and images the dipole dynamics of green fluorescent protein-labeled microtubules in live U2OS cells. pSIM applies directly to a large variety of commercial and home-built SIM systems with various imaging modality.

The folded mitochondria inner membrane-cristae is the structural foundation for oxidative phosphorylation (OXPHOS) and energy production. By mechanically simulating mitochondria morphogenesis, we speculate that efficient sculpting of the cristae is organelle non-autonomous. It has long been inferred that folding requires buckling in living systems. However, the tethering force for cristae formation and regulation has not been identified. Combining electron tomography, proteomics strategies, super resolution live cell imaging and mathematical modeling, we reveal that the mitochondria localized actin motor-myosin 19 (Myo19) is critical for maintaining cristae structure, by associating with the SAM-MICOS super complex. We discover that depletion of Myo19 or disruption of its motor activity leads to altered mitochondria membrane potential and decreased OXPHOS. We propose that Myo19 may act as a mechanical tether for effective ridging of the mitochondria cristae, thus sustaining the energy homeostasis essential for various cellular functions.

Class XIX myosin (Myo19) is a vertebrate-specific unconventional myosin, responsible for the transport of mitochondria. To characterize biochemical properties of Myo19, we prepared recombinant mouse Myo19-truncated constructs containing the motor domain and the IQ motifs using the baculovirus/Sf9 expression system. We identified regulatory light chain (RLC) of smooth muscle/non-muscle myosin-2 as the light chain of Myo19. The actin-activated ATPase activity and the actin-gliding velocity of Myo19-truncated constructs were about one-third and one-sixth as those of myosin-5a, respectively. The apparent affinity of Myo19 to actin was about the same as that of myosin-5a. The RLCs bound to Myo19 could be phosphorylated by myosin light chain kinase, but this phosphorylation had little effect on the actin-activated ATPase activity and the actin-gliding activity of Myo19-truncated constructs. Using dual fluorescence-labeled actin filaments, we determined that Myo19 is a plus-end-directed molecular motor. We found that, similar to that of the high-duty ratio myosin, such as myosin-5a, ADP release rate was comparable with the maximal actin-activated ATPase activity of Myo19, indicating that ADP release is a rate-limiting step for the ATPase cycle of acto-Myo19. ADP strongly inhibited the actin-activated ATPase activity and actin-gliding activity of Myo19-truncated constructs. Based on the above results, we concluded that Myo19 is a high-duty ratio molecular motor moving to the plus-end of the actin filament.