Nuclear transport of single molecules: dwell times at the nuclear pore complex.
AuthorsKubitscheck U, Grünwald D, Hoekstra A, Rohleder D, Kues T, Siebrasse JP, Peters R
JournalJ Cell Biol
PubMed ID15657394
'The mechanism by which macromolecules are selectively translocated through the nuclear pore complex (NPC) is still essentially unresolved. Single molecule methods can provide unique information on topographic properties and kinetic processes of asynchronous supramolecular assemblies with excellent spatial and time resolution. Here, single-molecule far-field fluorescence microscopy was applied to 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
Homotypic fusion of early endosomes: SNAREs do not determine fusion specificity.
AuthorsBrandhorst D, Zwilling D, Rizzoli SO, Lippert U, Lang T, Jahn R
JournalProc Natl Acad Sci U S A
PubMed ID16469845
'Membrane fusion in the secretory pathway is mediated by soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins. Different fusion steps are thought to be effected by independent sets of SNAREs, but it is unclear whether specificity is determined by an intrinsic specificity of SNARE pairing or by upstream factors. Using a ... More
Katanin is responsible for the M-phase microtubule-severing activity in Xenopus eggs.
AuthorsMcNally FJ, Thomas S
JournalMol Biol Cell
PubMed ID9658175
'Microtubules are dynamic structures whose proper rearrangement during the cell cycle is essential for the positioning of membranes during interphase and for chromosome segregation during mitosis. The previous discovery of a cyclin B/cdc2-activated microtubule-severing activity in M-phase Xenopus egg extracts suggested that a microtubule-severing protein might play an important role ... 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
Quantum-dot-assisted characterization of microtubule rotations during cargo transport.
AuthorsNitzsche B, Ruhnow F, Diez S,
JournalNat Nanotechnol
PubMed ID18772917
Owing to their wide spectrum of in vivo functions, motor proteins, such as kinesin-1, show great potential for application as nanomachines in engineered environments. When attached to a substrate surface, these motors are envisioned to shuttle cargo that is bound to reconstituted microtubules--one component of the cell cytoskeleton--from one location ... More
Pancreatic beta-cells secrete insulin in fast- and slow-release forms.
AuthorsMichael DJ, Ritzel RA, Haataja L, Chow RH,
JournalDiabetes
PubMed ID16505221
Insulin vesicles contain a chemically rich mixture of cargo that includes ions, small molecules, and proteins. At present, it is unclear if all components of this cargo escape from the vesicle at the same rate or to the same extent during exocytosis. Here, we demonstrate through real-time imaging that individual ... More
Actin restricts FcepsilonRI diffusion and facilitates antigen-induced receptor immobilization.
AuthorsAndrews NL, Lidke KA, Pfeiffer JR, Burns AR, Wilson BS, Oliver JM, Lidke DS,
JournalNat Cell Biol
PubMed ID18641640
The actin cytoskeleton has been implicated in restricting diffusion of plasma membrane components. Here, simultaneous observations of quantum dot-labelled FcepsilonRI motion and GFP-tagged actin dynamics provide direct evidence that actin filament bundles define micron-sized domains that confine mobile receptors. Dynamic reorganization of actin structures occurs over seconds, making the location ... 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
Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells.
AuthorsDouglass AD, Vale RD
JournalCell
PubMed ID15960980
Membrane subdomains have been implicated in T cell signaling, although their properties and mechanisms of formation remain controversial. Here, we have used single-molecule and scanning confocal imaging to characterize the behavior of GFP-tagged signaling proteins in Jurkat T cells. We show that the coreceptor CD2, the adaptor protein LAT, 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
Quantitative motion analysis of subchromosomal foci in living cells using four-dimensional microscopy
AuthorsBornfleth H, Edelmann P, Zink D, Cremer T, Cremer C
JournalBiophys J
PubMed ID10545385
The motion of subchromosomal foci and of whole chromosome territories in live human cell nuclei was investigated in four-dimensional space-time images. Visualization of subchromosomal foci was achieved by incorporating Cy3-dUTP into the nuclear DNA of two different cell types after microinjection. A subsequent segregation of the labeled cell nuclei led ... 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
High speed sCMOS-based oblique plane microscopy applied to the study of calcium dynamics in cardiac myocytes.
AuthorsSikkel MB, Kumar S, Maioli V, Rowlands C, Gordon F, Harding SE, Lyon AR, MacLeod KT, Dunsby C
JournalJ Biophotonics
PubMed ID26488431
'Oblique plane microscopy (OPM) is a form of light sheet microscopy that uses a single high numerical aperture microscope objective for both fluorescence excitation and collection. In this paper, measurements of the relative collection efficiency of OPM are presented. An OPM system incorporating two sCMOS cameras is then introduced that ... More
Open-source image reconstruction of super-resolution structured illumination microscopy data in ImageJ.
AuthorsMüller M, Mönkemöller V, Hennig S, Hübner W, Huser T
JournalNat Commun
PubMed ID26996201
Super-resolved structured illumination microscopy (SR-SIM) is an important tool for fluorescence microscopy. SR-SIM microscopes perform multiple image acquisitions with varying illumination patterns, and reconstruct them to a super-resolved image. In its most frequent, linear implementation, SR-SIM doubles the spatial resolution. The reconstruction is performed numerically on the acquired wide-field image ... More
Correlative Single-Molecule Localization Microscopy and Confocal Microscopy.
AuthorsSoeller C, Hou Y, Jayasinghe ID, Baddeley D, Crossman D
JournalMethods Mol Biol
PubMed ID28924670
Single-molecule localization microscopy allows the ability to image fluorescence labeled molecular targets at nanoscale resolution. However, for many biological questions the ability to provide tissue and cellular context in addition to these high resolution data is eminently informative. Here, we describe a procedure to achieve this aim by correlatively imaging ... More
Quantitative 3D structured illumination microscopy of nuclear structures.
AuthorsKraus F, Miron E, Demmerle J, Chitiashvili T, Budco A, Alle Q, Matsuda A, Leonhardt H, Schermelleh L, Markaki Y
JournalNat Protoc
PubMed ID28406495
3D structured illumination microscopy (3D-SIM) is the super-resolution technique of choice for multicolor volumetric imaging. Here we provide a validated sample preparation protocol for labeling nuclei of cultured mammalian cells, image acquisition and registration practices, and downstream image analysis of nuclear structures and epigenetic marks. Using immunostaining and replication labeling ... More
Super-Resolution Correlative Light and Electron Microscopy (SR-CLEM) Reveals Novel Ultrastructural Insights Into Dendritic Cell Podosomes.
AuthorsJoosten B, Willemse M, Fransen J, Cambi A, van den Dries K
JournalFront Immunol
PubMed ID30186284
Podosomes are multimolecular cytoskeletal structures that coordinate the migration of tissue-resident dendritic cells (DCs). They consist of a protrusive actin-rich core and an adhesive integrin-rich ring that contains adaptor proteins such as vinculin and zyxin. Individual podosomes are typically interconnected by a dense network of actin filaments giving rise to ... More
Three-Color Simultaneous Live Imaging of Autophagy-Related Structures.
AuthorsUeda H, Kunitaki O, Hamasaki M
JournalMethods Mol Biol
PubMed ID30610700
Simultaneous live cell imaging of multiple proteins helps to analyze mobility and interactions among proteins over time. Since autophagosomes depend on other organelles for their formation, it is necessary to observe this process with multiple fluorsphores to mark multiple organelles and the autophagosomes. To do so, we set up three cameras on one microscope ... More
Video-rate multi-color structured illumination microscopy with simultaneous real-time reconstruction.
AuthorsMarkwirth A, Lachetta M, Mönkemöller V, Heintzmann R, Hübner W, Huser T, Müller M
JournalNat Commun
PubMed ID31541134
Super-resolved structured illumination microscopy (SR-SIM) is among the fastest fluorescence microscopy techniques capable of surpassing the optical diffraction limit. Current custom-build instruments are able to deliver two-fold resolution enhancement with high acquisition speed. SR-SIM is usually a two-step process, with raw-data acquisition and subsequent, time-consuming post-processing for image reconstruction. In ... More