SUMMARY Lipid droplets (LDs) store metabolic energy and membrane lipid precursors. With excess metabolic energy, cells synthesize triacylglycerol (TG) and form LDs that grow dramatically. It is unclear how TG synthesis relates to LD formation and growth. Here, we identify two LD subpopulations: smaller LDs of relatively constant size, and LDs that grow larger. The latter population contains isoenzymes for each step of TG synthesis. Glycerol-3-phosphate acyltransferase 4 (GPAT4), which catalyzes the first and rate-limiting step, relocalizes from the endoplasmic reticulum (ER) to a subset of forming LDs, where it becomes stably associated. ER-to-LD targeting of GPAT4 and other LD-localized TG synthesis isozymes is required for LD growth. Key features of GPAT4 ER-to-LD targeting and function in LD growth are conserved between Drosophila and mammalian cells. Our results explain how TG synthesis is coupled with LD growth and identify two distinct LD subpopulations based on their capacity for localized TG synthesis.

Imaging volumes as thick as whole cells at three-dimensional (3D) super-resolution is required to reveal unknown features of cellular organization. We report a light microscope that generates images with translationally invariant 30 x 30 x 75 nm resolution over a depth of several micrometers. This method, named biplane (BP) FPALM, combines a double-plane detection scheme with fluorescence photoactivation localization microscopy (FPALM) enabling 3D sub-diffraction resolution without compromising speed or sensitivity.

Newly developed scientific complementary metal–oxide–semiconductor (sCMOS) cameras have the potential to dramatically accelerate data acquisition in single-molecule switching nanoscopy (SMSN) while simultaneously increasing the effective quantum efficiency. However, sCMOS-intrinsic pixel-dependent readout noise substantially reduces the localization precision and introduces localization artifacts. Here we present algorithms that overcome these limitations and provide unbiased, precise localization of single molecules at the theoretical limit. In combination with a multi-emitter fitting algorithm, we demonstrate single-molecule localization super-resolution imaging at up to 32 reconstructed images/second (recorded at 1,600–3,200 camera frames/second) in both fixed and living cells.

Methods based on single-molecule localization and photophysics have brought nanoscale imaging with visible light into reach. This has enabled single-particle tracking applications for studying the dynamics of molecules and nanoparticles and contributed to the recent revolution in super-resolution localization microscopy techniques. Crucial to the optimization of such methods are the precision and accuracy with which single fluorophores and nanoparticles can be localized. We present a lucid synthesis of the developments on this localization precision and accuracy and their practical implications in order to guide the increasing number of researchers using single-particle tracking and super-resolution localization microscopy.

The widely conserved ParABS system plays a major role in bacterial chromosome segregation. How the components of this system work together to generate translocation force and directional motion remains uncertain. Here, we combine biochemical approaches, quantitative imaging and mathematical modeling to examine the mechanism by which ParA drives the translocation of the ParB/parS partition complex in Caulobacter crescentus. Our experiments, together with simulations grounded on experimentally-determined biochemical and cellular parameters, suggest a novel 'DNA-relay' mechanism in which the chromosome plays a mechanical function. In this model, DNA-bound ParA-ATP dimers serve as transient tethers that harness the elastic dynamics of the chromosome to relay the partition complex from one DNA region to another across a ParA-ATP dimer gradient. Since ParA-like proteins are implicated in the partitioning of various cytoplasmic cargos, the conservation of their DNA-binding activity suggests that the DNA-relay mechanism may be a general form of intracellular transport in bacteria.DOI: http://dx.doi.org/10.7554/eLife.02758.001

Cytokinesis in animals, fungi, and amoebas depends on the constriction of a contractile ring built from a common set of conserved proteins. Many fundamental questions remain about how these proteins organize to generate the necessary tension for cytokinesis. Using quantitative high-speed fluorescence photoactivation localization microscopy (FPALM), we probed this question in live fission yeast cells at unprecedented resolution. We show that nodes, protein assembly precursors to the contractile ring, are discrete structural units with stoichiometric ratios and distinct distributions of constituent proteins. Anillin Mid1p, Fes/CIP4 homology-Bin/amphiphysin/ Rvs (F-BAR) Cdc15p, IQ motif containing GTPase-activating protein (IQGAP) Rng2p, and formin Cdc12p form the base of the node that anchors the ends of myosin II tails to the plasma membrane, with myosin II heads extending into the cytoplasm. This general node organization persists in the contractile ring where nodes move bidirectionally during constriction. We observed the dynamics of the actin network during cytokinesis, starting with the extension of short actin strands from nodes, which sometimes connected neighboring nodes. Later in cytokinesis, a broad network of thick bundles coalesced into a tight ring around the equator of the cell. The actin ring was ∼125 nm wide and ∼125 nm thick. These observations establish the organization of the proteins in the functional units of a cytokinetic contractile ring.T he mechanism of cell division by a contractile ring of actin and myosin II appeared in the common ancestor of amoebas, fungi, and animals (1). Although much is known about the protein composition of contractile rings, relatively little is known about the 3D organization of these proteins. This information is required to formulate computer models and simulations to test ideas regarding the mechanisms of contractile ring function.Electron microscopy has shown actin filaments in contractile rings of animal cells (2, 3) and yeast cells (4,5). Myosin II concentrates in cleavage furrow (6) and is the main motor for constricting contractile rings (7,8). In animal cells, electron microscopy has revealed rods the size of myosin II minifilaments in contractile rings (2, 9), and structured-illumination fluorescence microscopy has shown that this myosin II is organized in bipolar assemblies (10). Contractile rings contain other structural and regulatory proteins, including anillin (11), IQ motif containing GTPase-activating proteins (IQGAP) (12), formins (13), alphaactinin (14), and Fes/CIP4 homology-Bin/amphiphysin/Rvs (F-BAR) proteins (15), but how these proteins are anchored to the plasma membrane or organized into functional complexes in animal cells is unknown.Our understanding of the cytokinetic apparatus is most advanced in fission yeast (16). Only in fission yeast do we know the concentrations of the major cytokinesis proteins (17) and the time course of events leading to cellular division (18,19). Molecularly explicit computer models can account for both the ...

SummaryFluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50–80 nm) and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells. We demonstrate the wide applicability of W-4PiSMSN across diverse research fields by imaging complex molecular architectures ranging from bacteriophages to nuclear pores, cilia, and synaptonemal complexes in large 3D cellular volumes.

We describe a simple way to generate a wide-area high-resolution focus grid by in-line holography and study the factors that impacts its quality. In our holographic recording setup, the reference beam was the direct transmission of the incoming collimated laser beam through a mask coating with thin metal film, and the sample beam was the transmission of the laser through small apertures fabricated on the mask. The interference of the two beams was then recorded by a holographic plate positioned behind the mask. Compared with other recording schemes, the in-line holography scheme has many distinct advantages and is more suitable for generating a wide-area focus grid. We explored the dependence of diffraction quality, including reconstructed focus spot intensity and spot size, on different parameters for recording, such as optical density of the metal film, size of the apertures, and focal lengths. A wide-area focus grid (170 x 138 spots with area 5.1 mm x 4.1 mm) was recorded using the in-line holography scheme for a demonstration.