We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to È2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method-termed photoactivated localization microscopy-to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.

Starvation-induced autophagosomes engulf cytosol and/or organelles and deliver them to lysosomes for degradation, thereby re-supplying depleted nutrients. Despite advances in understanding the molecular basis of this process, the membrane origin of autophagosomes remains unclear. Here, we demonstrate that, in starved cells, autophagosomes are derived from the outer membranes of mitochondria. In time-lapse movies, the early autophagosomal marker, mApg5, transiently localizes to punctae on the surface of mitochondria, followed by the late autophagosomal marker, LC3. A unique tail-anchored outer mitochondrial membrane protein, but not other outer nor inner mitochondrial membrane proteins, labels autophagosomes and diffuses into newly forming autophagosomes from mitochondria. The fluorescent lipid, NBD-PS (which converts to PE in mitochondria) transfers from mitochondria to autophagosomes in starved cells. In addition, when mitochondria/ER connections are perturbed by loss of mitofusin2, starvation-induced autophagosomes do not form. Mitochondria thus play a central role in starvation-induced autophagy, serving as membrane source of autophagosomes.

Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.fluorescence microscopy ͉ interferometry ͉ PALM ͉ photoactivated localization microscopy ͉ single molecule imaging A fundamental question in biomedical research is how specific, nanometer-scale biomolecules are organized into multicomponent micron-scale structural and signaling ensembles that facilitate cell function. For example, microtubules are built of 8-nm tubulin subunits that incorporate on the ultrastructural level into polymers 25 nm in diameter and Ͼ10 m in length that serve as the building blocks of superstructures such as mitotic spindles and flagella. However, key challenges remain for determining cellular ultrastructure with high molecular specificity. Because cellular structures are organized on the nanoscale, nanometer resolution is required. Immunoelectron microscopy (EM)-based approaches provide the necessary resolution, but they lack robust molecular specificity because the large size of the antibodies hampers their penetration into dense structures and the specificity of the antibody can be compromised by cross-reactivity and epitope masking caused by the harsh fixation often used for high-resolution EM. Fluorescence microscopy coupled with fluorescent protein (FP) fusion technology enables imaging cellular structure with exquisite molecular specificity, but the resolution of 3D images is diffraction-limited to Ϸ200 nm in the lateral and Ϸ500 nm in the axial direction, limiting conventional fluorescence to the characterization of cellular superstructure. Some of the recent fluorescence-based superresolution microscopy techniques (1-5) demonstrated a resolution of Ͻ100 nm in the vertical direction; however, this is still insufficient to bridge the resolution gap between cellular ultrastructure and superstructure. To achieve near-ultrastructural 3D resolution even for the limited photon outputs of highmolecular-specificity FPs, we have developed a single-photon multiphase interferometric scheme and integrated it with a lateral photoactivated localization microscopy (PALM) (6

The final stage of cytokinesis is abscission, the cutting of the narrow membrane bridge connecting two daughter cells. The endosomal sorting complex required for transport (ESCRT) machinery is required for cytokinesis, and ESCRT-III has membrane scission activity in vitro, but the role of ESCRTs in abscission has been undefined. Here, we use structured illumination microscopy and timelapse imaging to dissect the behavior of ESCRTs during abscission. Our data reveal that the ESCRT-I subunit tumor-susceptibility gene 101 (TSG101) and the ESCRT-III subunit charged multivesicular body protein 4b (CHMP4B) are sequentially recruited to the center of the intercellular bridge, forming a series of cortical rings. Late in cytokinesis, however, CHMP4B is acutely recruited to the narrow constriction site where abscission occurs. The ESCRT disassembly factor vacuolar protein sorting 4 (VPS4) follows CHMP4B to this site, and cell separation occurs immediately. That arrival of ESCRT-III and VPS4 correlates both spatially and temporally with the abscission event suggests a direct role for these proteins in cytokinetic membrane abscission.