An optimized method for 3D fluorescence co-localization applied to human kinetochore protein architecture.

Two-color fluorescence co-localization in 3D (three-dimension) has the potential to achieve accurate measurements at the nanometer length scale. Here, we optimized a 3D fluorescence co-localization method that uses mean values for chromatic aberration correction to yield the mean separation with ~10 nm accuracy between green and red fluorescently labeled protein epitopes within single human kinetochores. Accuracy depended critically on achieving small standard deviations in fluorescence centroid determination, chromatic aberration across the measurement field, and coverslip thickness. Computer simulations showed that large standard deviations in these parameters significantly increase 3D measurements from their true values. Our 3D results show that at metaphase, the protein linkage between CENP-A within the inner kinetochore and the microtubule-binding domain of the Ndc80 complex within the outer kinetochore is on average ~90 nm. The Ndc80 complex appears fully extended at metaphase and exhibits the same subunit structure in vivo as found in vitro by crystallography.

A Kinesin-5, Cin8, Recruits Protein Phosphatase 1 to Kinetochores and Regulates Chromosome Segregation.

Kinesin-5 is a highly conserved homo-tetrameric protein complex responsible for crosslinking microtubules and pushing spindle poles apart. The budding yeast Kinesin-5, Cin8, is highly concentrated at kinetochores in mitosis before anaphase, but its functions there are largely unsolved. Here, we show that Cin8 localizes to kinetochores in a cell-cycle-dependent manner and concentrates near the microtubule binding domains of Ndc80 at metaphase. Cin8's kinetochore localization depends on the Ndc80 complex, kinetochore microtubules, and the Dam1 complex. Consistent with its kinetochore localization, a Cin8 deletion induces a loss of tension at the Ndc80 microtubule binding domains and a major delay in mitotic progression. Cin8 associates with Protein Phosphatase 1 (PP1), and mutants that inhibit its PP1 binding also induce a loss of tension at the Ndc80 microtubule binding domains and delay mitotic progression. Taken together, our results suggest that Cin8-PP1 plays a critical role at kinetochores to promote accurate chromosome segregation by controlling Ndc80 attachment to microtubules.

Morphometric data on severely and morbidly obese deceased, established on forensic and non-forensic autopsies.

With the widespread increase in the incidence of obesity, autopsies on severely and morbidly obese deceased have become common in the USA. Standard reference tables for organ weights provide little or no information on individuals with a body mass index greater than 35 kg/m(2). Although several recent reports have provided organ weights for small numbers of morbidly obese persons who died naturally from a variety of causes, these data may have been affected by comorbidities. Furthermore, they did not provide information relative to differences in organ weight based on gender, age, and race. The aim of the present study was to fill this void by developing reference tables for organ weights of severely and morbidly obese individuals. Our study was based on data from 802 forensic and medical autopsies, including 435 cases of death of natural and 367 of non-natural causes. Organ weights were compared between these groups, and reference ranges were generated. Significant variability was found in organ weights especially among deceased older than 40 years who died naturally, suggesting that comorbidities affect organ weight. Reference tables were compiled for organ weights and morphometric data based on gender, age, and race. Since obesity is a pathological condition affecting organ weight, these reference tables do not reflect normal organ weights but only weight as seen in severely and morbidly obese individuals. They should be useful to pathologists who perform forensic and non-forensic autopsies.

A comparison of midline and tracheal gene regulation during Drosophila development.

Within the Drosophila embryo, two related bHLH-PAS proteins, Single-minded and Trachealess, control development of the central nervous system midline and the trachea, respectively. These two proteins are bHLH-PAS transcription factors and independently form heterodimers with another bHLH-PAS protein, Tango. During early embryogenesis, expression of Single-minded is restricted to the midline and Trachealess to the trachea and salivary glands, whereas Tango is ubiquitously expressed. Both Single-minded/Tango and Trachealess/Tango heterodimers bind to the same DNA sequence, called the CNS midline element (CME) within cis-regulatory sequences of downstream target genes. While Single-minded/Tango and Trachealess/Tango activate some of the same genes in their respective tissues during embryogenesis, they also activate a number of different genes restricted to only certain tissues. The goal of this research is to understand how these two related heterodimers bind different enhancers to activate different genes, thereby regulating the development of functionally diverse tissues. Existing data indicates that Single-minded and Trachealess may bind to different co-factors restricted to various tissues, causing them to interact with the CME only within certain sequence contexts. This would lead to the activation of different target genes in different cell types. To understand how the context surrounding the CME is recognized by different bHLH-PAS heterodimers and their co-factors, we identified and analyzed novel enhancers that drive midline and/or tracheal expression and compared them to previously characterized enhancers. In addition, we tested expression of synthetic reporter genes containing the CME flanked by different sequences. Taken together, these experiments identify elements overrepresented within midline and tracheal enhancers and suggest that sequences immediately surrounding a CME help dictate whether a gene is expressed in the midline or trachea.