Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging.
AuthorsSzymborska A, de Marco A, Daigle N, Cordes VC, Briggs JA, Ellenberg J,
Journal
PubMed ID23845946
'Much of life''s essential molecular machinery consists of large protein assemblies that currently pose challenges for structure determination. A prominent example is the nuclear pore complex (NPC), for which the organization of its individual components remains unknown. By combining stochastic super-resolution microscopy, to directly resolve the ringlike structure of the ... More
The cohesion protein ORD is required for homologue bias during meiotic recombination.
AuthorsWebber HA, Howard L, Bickel SE
JournalJ Cell Biol
PubMed ID15007062
'During meiosis, sister chromatid cohesion is required for normal levels of homologous recombination, although how cohesion regulates exchange is not understood. Null mutations in orientation disruptor (ord) ablate arm and centromeric cohesion during Drosophila meiosis and severely reduce homologous crossovers in mutant oocytes. We show that ORD protein localizes along ... More
Exocytosis of IgG as mediated by the receptor, FcRn: an analysis at the single-molecule level.
AuthorsOber RJ, Martinez C, Lai X, Zhou J, Ward ES
JournalProc Natl Acad Sci U S A
PubMed ID15258288
'IgG transport within and across cells is essential for effective humoral immunity. Through a combination of biochemical and in vivo analyses, the MHC class I-related neonatal Fc receptor (FcRn) is known to play a central role in delivering IgGs within and across cells. However, little is known about the molecular ... More
Fast, three-dimensional super-resolution imaging of live cells.
AuthorsJones SA, Shim SH, He J, Zhuang X,
JournalNat Methods
PubMed ID21552254
'We report super-resolution fluorescence imaging of live cells with high spatiotemporal resolution using stochastic optical reconstruction microscopy (STORM). By labeling proteins either directly or via SNAP tags with photoswitchable dyes, we obtained two-dimensional (2D) and 3D super-resolution images of living cells, using clathrin-coated pits and the transferrin cargo as model ... More
Practical confocal microscopy and the evaluation of system performance.
AuthorsZucker RM, Price OT
JournalMethods
PubMed ID10491274
'The laser scanning confocal microscope has enormous potential in many fields of biology. Currently there is a subjective nature in the assessment of a confocal microscope''s performance by primarily evaluating the system with a specific test slide provided by the user''s laboratory. To achieve better performance from the equipment, it ... More
AuthorsPuri N, Kruhlak MJ, Whiteheart SW, Roche PA
JournalJ Immunol
PubMed ID14607937
'Mast cells possess specialized granules that, upon stimulation of surface FcR with IgE, fuse with the plasma membrane, thereby releasing inflammatory mediators. A family of membrane fusion proteins called SNAREs, which are present on both the granule and the plasma membrane, plays a role in the fusion of these granules ... More
High accuracy 3D quantum dot tracking with multifocal plane microscopy for the study of fast intracellular dynamics in live cells.
AuthorsRam S, Prabhat P, Chao J, Ward ES, Ober RJ,
JournalBiophys J
PubMed ID18835896
'Single particle tracking in three dimensions in a live cell environment holds the promise of revealing important new biological insights. However, conventional microscopy-based imaging techniques are not well suited for fast three-dimensional (3D) tracking of single particles in cells. Previously we developed an imaging modality multifocal plane microscopy (MUM) to ... More
Correlative light microscopy for high-content screening.
AuthorsFlottmann B, Gunkel M, Lisauskas T, Heilemann M, Starkuviene V, Reymann J, Erfle H,
Journal
PubMed ID24215639
High-throughput microscopy is an effective tool for rapidly collecting data on a large scale. However, high throughput comes at the cost of low spatial resolution. Here we introduce correlative light microscopy by combining fast automated widefield imaging, confocal microscopy and super-resolution microscopy. We demonstrate the potential of this approach for ... More
Sample preparation for single molecule localization microscopy.
AuthorsAllen JR, Ross ST, Davidson MW,
Journal
PubMed ID24084850
Single molecule localization-based optical nanoscopy was introduced in 2006, surpassing traditional diffraction-limited resolutions by an order of magnitude. Seven years later, this superresolution technique is continuing to follow a trend of increasing popularity and pervasiveness, with the proof-of-concept work long finished and commercial implementations now available. However one important aspect ... More
Widely accessible method for superresolution fluorescence imaging of living systems.
AuthorsDedecker P, Mo GC, Dertinger T, Zhang J,
JournalProc Natl Acad Sci U S A
PubMed ID22711840
Superresolution fluorescence microscopy overcomes the diffraction resolution barrier and allows the molecular intricacies of life to be revealed with greatly enhanced detail. However, many current superresolution techniques still face limitations and their implementation is typically associated with a steep learning curve. Patterned illumination-based superresolution techniques [e.g., stimulated emission depletion (STED), ... More
Test samples for optimizing STORM super-resolution microscopy.
AuthorsMetcalf DJ, Edwards R, Kumarswami N, Knight AE,
Journal
PubMed ID24056752
STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize ... More
Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy.
Fluorescence light microscopy allows multicolor visualization of cellular components with high specificity, but its utility has until recently been constrained by the intrinsic limit of spatial resolution. We applied three-dimensional structured illumination microscopy (3D-SIM) to circumvent this limit and to study the mammalian nucleus. By simultaneously imaging chromatin, nuclear lamina, ... More
Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes.
AuthorsShim SH, Xia C, Zhong G, Babcock HP, Vaughan JC, Huang B, Wang X, Xu C, Bi GQ, Zhuang X,
JournalProc Natl Acad Sci U S A
PubMed ID22891300
Imaging membranes in live cells with nanometer-scale resolution promises to reveal ultrastructural dynamics of organelles that are essential for cellular functions. In this work, we identified photoswitchable membrane probes and obtained super-resolution fluorescence images of cellular membranes. We demonstrated the photoswitching capabilities of eight commonly used membrane probes, each specific ... More
Cytogenetic evidence for asexual evolution of bdelloid rotifers.
AuthorsMark Welch JL, Mark Welch DB, Meselson M
JournalProc Natl Acad Sci U S A
PubMed ID14747655
DNA sequencing has shown individual bdelloid rotifer genomes to contain two or more diverged copies of every gene examined and has revealed no closely similar copies. These and other findings are consistent with long-term asexual evolution of bdelloids. It is not entirely ruled out, however, that bdelloid genomes consist of ... More
The mitotic DNA damage checkpoint proteins Rad17 and Rad24 are required for repair of double-strand breaks during meiosis in yeast.
AuthorsShinohara M, Sakai K, Ogawa T, Shinohara A
JournalGenetics
PubMed ID12871899
We show here that deletion of the DNA damage checkpoint genes RAD17 and RAD24 in Saccharomyces cerevisiae delays repair of meiotic double-strand breaks (DSBs) and results in an altered ratio of crossover-to-noncrossover products. These mutations also decrease the colocalization of immunostaining foci of the RecA homologs Rad51 and Dmc1 and ... More
Determinants of liposome fusion mediated by synaptic SNARE proteins.
AuthorsSchuette CG, Hatsuzawa K, Margittai M, Stein A, Riedel D, Küster P, König M, Seidel C, Jahn R
JournalProc Natl Acad Sci U S A
PubMed ID14981239
Synaptic exocytosis requires the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin 1, SNAP-25, and synaptobrevin (VAMP). Assembly of the SNAREs into a stable core complex is supposed to catalyze membrane fusion, and proteoliposomes reconstituted with synaptic SNARE proteins spontaneously fuse with each other. We now show that liposome ... More
The Saccharomyces cerevisiae spindle pole body is a dynamic structure.
AuthorsYoder TJ, Pearson CG, Bloom K, Davis TN
JournalMol Biol Cell
PubMed ID12925780
During spindle pole body (SPB) duplication, the new SPB is assembled at a distinct site adjacent to the old SPB. Using quantitative fluorescence methods, we studied the assembly and dynamics of the core structural SPB component Spc110p. The SPB core exhibits both exchange and growth in a cell cycle-dependent manner. ... More
Localization of a putative transcriptional regulator (ATRX) at pericentromeric heterochromatin and the short arms of acrocentric chromosomes.
ATRX is a member of the SNF2 family of helicase/ATPases that is thought to regulate gene expression via an effect on chromatin structure and/or function. Mutations in the hATRX gene cause severe syndromal mental retardation associated with alpha-thalassemia. Using indirect immunofluorescence and confocal microscopy we have shown that ATRX protein ... More
Epitope-dependent localization of estrogen receptor-alpha, but not -beta, in en face arterial endothelium.
AuthorsDan P, Cheung JC, Scriven DR, Moore ED
JournalAm J Physiol Heart Circ Physiol
PubMed ID12531733
Rapid, nongenomic effects of 17 beta-estradiol (E(2)) in endothelial cells are postulated to arise from membrane-associated estrogen receptors (ERs), which have not been visualized in vascular tissue. To identify membrane ERs, we used multiple site-directed ER alpha or ER beta antibodies to label en face rat cerebral and coronary arterial ... More
Meiotic cohesion requires accumulation of ORD on chromosomes before condensation.
AuthorsBalicky EM, Endres MW, Lai C, Bickel SE
JournalMol Biol Cell
PubMed ID12429833
Cohesion between sister chromatids is a prerequisite for accurate chromosome segregation during mitosis and meiosis. To allow chromosome condensation during prophase, the connections that hold sister chromatids together must be maintained but still permit extensive chromatin compaction. In Drosophila, null mutations in the orientation disruptor (ord) gene lead to meiotic ... More
Site of docking and fusion of insulin secretory granules in live MIN6 beta cells analyzed by TAT-conjugated anti-syntaxin 1 antibody and total internal reflection fluorescence microscopy.
AuthorsOhara-Imaizumi M, Nishiwaki C, Kikuta T, Kumakura K, Nakamichi Y, Nagamatsu S
JournalJ Biol Chem
PubMed ID14676208
To determine the site of insulin exocytosis in the pancreatic beta cell plasma membrane, we analyzed the interaction between the docking/fusion of green fluorescent protein-tagged insulin granules and syntaxin 1 labeled by TAT-conjugated Cy3-labeled antibody (Ab) using total internal reflection fluorescence microscopy (TIRFM). Monoclonal Ab against syntaxin 1 was labeled ... More
Specific localization of serine 19 phosphorylated myosin II during cell locomotion and mitosis of cultured cells.
AuthorsMatsumura F, Ono S, Yamakita Y, Totsukawa G, Yamashiro S
JournalJ Cell Biol
PubMed ID9425160
Phosphorylation of the regulatory light chain of myosin II (RMLC) at Serine 19 by a specific enzyme, MLC kinase, is believed to control the contractility of actomyosin in smooth muscle and vertebrate nonmuscle cells. To examine how such phosphorylation is regulated in space and time within cells during coordinated cell ... More
Precise tracking of the dynamics of multiple proteins in endocytic events.
AuthorsPicco A, Kaksonen M
JournalMethods Cell Biol
PubMed ID28215339
'Endocytosis is a complex and dynamic process that involves dozens of different proteins to define the site of endocytosis, form a membrane invagination, and pinch off a membrane vesicle into the cytoplasm. Fluorescent light microscopy is a powerful tool to visualize the dynamic behaviors of the proteins taking part in ... More
New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography.
AuthorsSchorb M, Gaechter L, Avinoam O, Sieckmann F, Clarke M, Bebeacua C, Bykov YS, Sonnen AF, Lihl R, Briggs JAG
JournalJ Struct Biol
PubMed ID27368127
'Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare dynamic events or specific objects can be identified, targeted and imaged by electron microscopy or tomography. To combine it with structural studies using cryo-electron microscopy ... More
A Method to Visualize the Nanoscopic Morphology of Astrocytes In Vitro and In Situ.
AuthorsHeller JP, Rusakov DA
JournalMethods Mol Biol
PubMed ID30617973
'In recent years it has become apparent that astroglia are not only essential players in brain development, homeostasis, and metabolic support but are also important for the formation and regulation of synaptic circuits. Fine astrocytic processes that can be found in the vicinity of synapses undergo considerable structural plasticity associated with ... More
Quantitative Analysis of Nuclear Lamins Imaged by Super-Resolution Light Microscopy.
AuthorsKittisopikul M, Virtanen L, Taimen P, Goldman RD
JournalCells
PubMed ID31003483
'The nuclear lamina consists of a dense fibrous meshwork of nuclear lamins, Type V intermediate filaments, and is ~14 nm thick according to recent cryo-electron tomography studies. Recent advances in light microscopy have extended the resolution to a scale allowing for the fine structure of the lamina to be imaged ... More
Dual-Color and 3D Super-Resolution Microscopy of Multi-protein Assemblies.
AuthorsHoess P, Mund M, Reitberger M, Ries J
JournalMethods Mol Biol
PubMed ID29605918
Breaking the resolution limit of conventional microscopy by super-resolution microscopy (SRM) led to many new biological insights into protein assemblies at the nanoscale. Here we provide detailed protocols for single-molecule localization microscopy (SMLM) to image the structure of a protein complex. As examples, we show how to acquire single- and ... More
Imaging of Transcription and Replication in the Bacterial Chromosome with Multicolor Three-Dimensional Superresolution Structured Illumination Microscopy.
AuthorsMartin CM, Cagliero C, Sun Z, Chen Jin DJ
JournalMethods Mol Biol
PubMed ID30109608
Superresolution imaging technology has contributed to our understanding of the subnucleoid organization in E. coli cells. Multicolor superresolution images revealing "bacterial nucleolus-like structure or organization," "nucleolus-like compartmentalization of the transcription factories," and "spatial segregation of the transcription and replication machineries" have enhanced our understanding of the dynamic landscape of the ... More