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To define the molecular architecture of the kinetochore in vertebrate cells, we measured the copy number of eight kinetochore proteins that link kinetochore microtubules (MTs [kMTs]) to centromeric DNA. We used a fluorescence ratio method and chicken DT40 cell lines in which endogenous loci encoding the analyzed proteins were deleted and complemented using integrated green fluorescent protein fusion transgenes. For a mean of 4.3 kMTs at metaphase, the protein copy number per kMT is between seven and nine for members of the MT-binding KNL-1/Mis12 complex/Ndc80 complex network. It was between six and nine for four members of the constitutive centromere-associated network: centromere protein C (CENP-C), CENP-H, CENP-I, and CENP-T. The similarity in copy number per kMT for all of these proteins suggests that each MT end is linked to DNA by six to nine fibrous unit attachment modules in vertebrate cells, a conclusion that indicates architectural conservation between multiple MT-binding vertebrate and single MT-binding budding yeast kinetochores. doi/10.1083/jcb.200912022

Total internal reflection fluorescence microscopy (TIRFM) images the plasma membrane--cytosol interface and has allowed insights into the behavior of individual secretory granules before and during exocytosis. Much less is known about the dynamics of the other partner in exocytosis, the plasma membrane. In this study, we report the implementation of a TIRFM-based polarization technique to detect rapid submicrometer changes in plasma membrane topology as a result of exocytosis. A theoretical analysis of the technique is presented together with image simulations of predicted topologies of the post-fusion granule membrane--plasma membrane complex. Experiments on dil-stained bovine adrenal chromaffin cells using polarized TIRFM demonstrate rapid and varied submicrometer changes in plasma membrane topology at sites of exocytosis that occur immediately upon fusion. We provide direct evidence for a persistent curvature in the exocytotic region that is altered by inhibition of dynamin guanosine triphosphatase activity and is temporally distinct from endocytosis measured by VMAT2-pHluorin. doi/10.1083/jcb.200908010

Tetraspanins regulate cell migration, sperm-egg fusion, and viral infection. Through interactions with one another and other cell surface proteins, tetraspanins form a network of molecular interactions called the tetraspanin web. In this study, we use single-molecule fluorescence microscopy to dissect dynamics and partitioning of the tetraspanin CD9. We show that lateral mobility of CD9 in the plasma membrane is regulated by at least two modes of interaction that each exhibit specific dynamics. The majority of CD9 molecules display Brownian behavior but can be transiently confined to an interaction platform that is in permanent exchange with the rest of the membrane. These platforms, which are enriched in CD9 and its binding partners, are constant in shape and localization. Two CD9 molecules undergoing Brownian trajectories can also codiffuse, revealing extra platform interactions. CD9 mobility and partitioning are both dependent on its palmitoylation and plasma membrane cholesterol. Our data show the high dynamic of interactions in the tetraspanin web and further indicate that the tetraspanin web is distinct from raft microdomains.

Membrane trafficking during cytokinesis is not well understood. We used advanced live cell imaging techniques to track exocytosis of single vesicles to determine whether constitutively exocytosed membrane is focally delivered to the cleavage furrow. Ultrasensitive three-dimensional confocal time-lapse imaging of the temperature-sensitive membrane cargo protein vesicular stomatitis virus protein--yellow fluorescent protein revealed that vesicles from both daughter cells traffic out of the Golgi and into the furrow, following curvilinear paths. Immunolocalization and photobleaching experiments indicate that individual vesicles accumulate at the midbody and generate a reserve vesicle pool that is distinct from endosomal and lysosomal compartments. Total internal reflection fluorescence microscopy imaging provided direct evidence that Golgi-derived vesicles from both daughter cells not only traffic to the furrow region but dock and fuse there, supporting a symmetrically polarized exocytic delivery model. In contrast, quantitative analysis of midbody abscission showed inheritance of the midbody remnant by one daughter cell, indicating that cytokinesis is composed of both symmetrical and asymmetrical stages.

Discrete localized fluorescence transients due to openings of a single plasma membrane Ca(super 2+) permeable cation channel were recorded using wide-field digital imaging microscopy with fluo-3 as the Ca(super 2+) indicator. These transients were obtained while simultaneously recording the unitary channel currents using the whole-cell current-recording configuration of the patch-clamp technique. This cation channel in smooth muscle cells is opened by caffeine (Guerrero, A., F.S. Fay, and J.J. Singer. 1994. J. Gen. Physiol. 104:375-394). The localized fluorescence transients appeared to occur at random locations on the cell membrane, with the duration of the rising phase matching the duration of the channel opening. Moreover, these transients were only observed in the presence of sufficient extracellular Ca(super 2+), suggesting that they are due to Ca(super 2+) influx from the bathing solution. The fluorescence transient is characterized by an initial fast rising phase when the channel opens, followed by a slower rising phase during prolonged openings. When the channel closes there is an immediate fast falling phase followed by a slower falling phase. Computer simulations of the underlying events were used to interpret the time course of the transients. The rapid phases are mainly due to the establishment or removal of Ca(super 2+) and Ca(super 2+)-bound fluo-3 gradients near the channel when the channel opens or closes, while the slow phases are due to the diffusion of Ca(super 2+) and Ca(super 2+)-bound fluo-3 into the cytoplasm. Transients due to short channel openings have a 'Ca(super 2+) spark-like' appearance, suggesting that the rising and early falling components of sparks (due to openings of ryanodine receptors) reflect the fast phases of the fluorescence change. The results presented here suggest methods to determine the relationship between the fluorescence transient and the underlying Ca(super 2+) current, to study intracellular localized Ca(super 2+) handling as might occur from single Ca(super 2+) channel openings, and to localize Ca(super 2+) permeable ion channels on the plasma membrane. Keywords: calcium; caffeine-activated channel; nonselective cation channels; fluo-3 fluorescence; smooth muscle

Research was conducted to determine the partial structure of the yeast La protein and compare it with human protein. Structural analysis indicates that the domain of 113-amino acids, involved in the RNA-binding, differs from human La.

The method for the detection of localized calcium ion (Ca2+) concentration by epifluorescence microscopy was improved by modifying the nearest neighbors deblurring scheme toobtain ratiometric fura-2 images from mast cells. It was observed that the restored image is almost the same as that obtained with the nearest neighbors scheme when the in focus image is used in lieu of adjacent adjacent image planes for the deblurring. The improved method allows improved measurements of cytosolic-free Ca2+, which is involved in the regulation of several important biological processes.

The translocation of fluorescently tagged PLC[Gamma] and requirements for this process in cells stimulated with EGF were analyzed using real time fluorescence microscopy applied for the first time to monitor growth factor receptor--effector interactions. The translocation of PLC[Gamma] to the plasma membrane required the functional Src homology 2 domains and was not affected by mutations in the pleckstrin homology domain or inhibition of phosphatidylinositol (PI) 3-kinase. An array of domains specific for PLC[Gamma] isoforms was sufficient for this translocation. The dynamics of translocation to the plasma membrane and redistribution of PLC[Gamma], relative to localization of the EGF receptor and PI 4,5-biphosphate (PI 4,5-[P.sub.2]), were shown. Colocalization with the receptor was observed in the plasma membrane and in membrane ruffles where PI 4,5-[P.sub.2] substrate could also be visualized. At later times, internalization of PLC[Gamma], which could lead to separation from the substrate, was observed. The data support a direct binding of PLC[Gamma] to the receptor as the main site of the plasma membrane recruitment. The presence of PLC[Gamma] in membrane structures and its access to the substrate appear to be transient and are followed by a rapid incorporation into intracellular vesicles, leading to downregulation of the PLC activity. Key words: PLC * EGF receptor * translocation * real time imaging * SH2 and PH domains