Robert Josephs - US grants

DESCRIPTION: (Adapted from investigator's abstract) This proposal is aimed at studying the molecular events involved in sickle cell hemoglobin polymerization and the consequent red cell deformation resulting from it. The ultimate goal of the project is to apply the information obtained from the studies to the amelioration of painful crises in sickle cell anemia patients. Beyond this practical application, the studies should offer insight into other systems involving macromolecular assemblies. Hb S polymerization proceeds by two distinct mechanisms: the first is a nucleation event leading to initial polymerization, and the second is an exponential increase in the rate of fiber formation as newly created surfaces nucleate additional fibers. Although the molecular contacts within fibers are known, little or nothing is known about the interfiber contacts or of the mechanisms involved in aggregation of fibers. Fifty- five mutants have been prepared so far and 33 affect fiber formation. Thirty-one occur at calculated intermolecular sites, and radial and linear growth are different. Thus, crystal coordinates by themselves have not allowed a full description of fiber propagation. Still less is known about the nature and formation of "irreversibly sickled cells", although their existence has been known for more than 20 years. These cells are rigid and retain a sickled shape, even in the absence of fibers. How the cytoskeleton of the cell has been altered to produce this peculiar situation is not known, but perhaps the sensitivity of skeletal proteins (especially spectrin) to ionic conditions may provide a clue. This proposal has two overall goals: study of the structure of fiber bundles by negative staining and cryoelectron microscopy, and the preparation of site- directed hemoglobin mutants to explore how specific substitutions affect fiber formation. The mutants will be studied by electron microscopy and the polymerization kinetics will be evaluated by video-enhanced light microscopy. These molecular events will be correlated with cellular alterations using cryomicroscopy to avoid perturbations of the skeletal structures caused by staining. The proposal involves four specific aims: 1. The mechanism of polymerization. Fiber bundles will be studied using negative staining cryoelectron microscopy in order to determine intermolecular contacts that facilitate nucleation of new fibers on the surface of existing fibers. 2. Site directed mutagenesis. Double mutants of hemoglobin S will be prepared to determine the effect of specific alterations on fiber formation. 3. Studies of fibers in high concentrations of phosphate buffer. This section of the work will attempt to determine the physiological relevance of previous data collected in high phosphate. 4. The role of the red cell cytoskeleton in cell deformation. Cryomicroscopy will be used to study the fine structure of the cytoskeleton of irreversibly sickled cells.

A group of investigators working on biological structures seeks support to purchase an electron microscope for a University multiuser facility. The electron microscope will be used for studies of the structures of sickle hemoglobin polymers, the acetycholine receptor, the red blood cell cytoskeleton, the eryth- ropoietin receptor, cardiac gap junctions, and nucleoprotein complexes. These studies require a very high resolution microscope equipped with a goniometer stage and capable of cryo and low dose microscopy. The applicant group of investigators are now entirely dependent upon a single twenty-one year old Philips EM 300 for high resolution work. Even though this microscope is well maintained, age-related problems are resulting in ever increasing difficulties. The proposed new microscope would be installed in an existing EM lab which contains all the supporting equipment normally found in electron microscope laboratories. This lab is staffed by a very competent electron microscope technician. The laboratory has very extensive computer facilities for digital image processing which complement the EM hardware. All of this existing infrastructure and equipment would be available to the user group. The lab has maintained (and will continue to maintain) a policy of providing access to the EM facility for research and training for all who wish to use it.

The goal of this work is to study the structure and assembly of polymers of sickle cell hemoglobin which are responsible for sickle cell anemia. These studies will involve electron microscopy of polymers in various stages of assembly. Rapid freezing in liquid ethane will be used to arrest the assembly process and the partially assembled polymers will be examined in the electron microscope. In a similar vein, studies of polymer disassembly will also be carried out in order to determine how this process occurs. The interpretation of these studies will draw heavily from existing models of the heterogeneous polymerization of HbS. A prime objective of the work is to identify the physical structures which mediate the association and dissociation reactions. Studies of the molecular structure of the fiber have been carried as far as seems possible using negative staining. We propose to extend these studies to higher resolution using cryomicroscopy. This technique involves rapidly freezing specimens in a thin layer of vitreous ice. Micrographs record the electron density of the specimen. This technique will allow us to extend the resolution of micrographs of HbS polymers from the existing 32A limit to 15A or better. This increase in resolution will provide a far more detailed image of the molecular structure of sickle hemoglobin polymers than has been possible to obtain up to now. These higher resolution data, taken in conjunction with the known atomic structure of HbS should permit mapping many of the intermolecular contacts which stabilize these clinically important structures.

DESCRIPTION (provided by applicant): The intracellular polymerization of sickle cell hemoglobin to form rod-like fibers causes sickle cell anemia. Cells containing fibers become distorted and rigid and occlude small capillaries causing tissue destruction. The structure of the fibers is of interest since this information could provide a rational basis for therapies aimed at inhibiting fiber formation. In addition, understanding the structural basis of HbS polymerization offers insights relevant to other systems involving macromolecular assemblies. We have succeeded in carrying out reconstructions of electron micrographs of frozen-hydrated HbS fibers. By averaging 10 fibers into the reconstruction we have improved the resolution of the reconstruction from 32A (previously published) to 16A radially and 8A axially. The improvement in resolution is caused by increasing the signal to noise ratio. We believe that the frozen-hydrated fibers are sufficiently well preserved that we could attain a resolution of at least 4 -6A by averaging several hundred particles into the reconstruction. A second series of experiments involves synthesis of double mutants (the HbS site and one other site) to explore how changes in individual amino acids effect the fiber properties. Site directed mutants at intermolecular contacts provide a means of critically testing current molecular models of the fiber structure by their effect on polymerization. We propose to study the structure of fibers containing the site directed mutants by electron microscopy and polymerization kinetics by video enhanced light microscopy.

A group of seven investigators will purchase a microdensitometer which will be incorporated in the Electron Microscopy and Image Processing Core Laboratory in the Cummings Life Science Center of the University of Chicago. The densitometer will be used in studies of the structure of sickle cell hemoglobin fibers, the structure of Dictoyostelium discoideum membrane, DNA protein complexes, the structure and mechanism of action of heat shock proteins, the structure of lipoproteins and the structure of secretory granules in Tetrahymena.

With support from the Major Research Instrumentation (MRI) Program, Norbert F. Scherer and colleagues at the University of Chicago will acquire a high-resolution scanning transmission electron microscope (S/TEM) for interdisciplinary nanoscience research. The STEM will be used to study systems in nano-physics, nano-chemistry, and nano-biology. Some of the applications include characterization of nanocrystals; diblock copolymer used for making nanoparticle array wires; phase separation and structure studies for electroactive diblock copolymers; hemoglobin fiber formation and sickle cell anemia; genome-scale genetic analysis and fabrication of nano-structured materials with novel properties; biomembranes; high resolution e-beam writing using photoresists; and new direct methods of pattern formation.

Much of the research at the forefront of structural and cell biology and nanoscience requires routine characterization of specimens by a combination of transmission electron microscopy (TEM) and scanning transmission electron microscopy (S/TEM). High-end TEM is unsurpassed in terms of image resolution and ease of interpretation. In addition, with new capabilities such as tomography, electron microscopy is entering a new era in which its application can visualize molecular structure with (near-) atomic resolution.

This award partially supports the acquisition of a high-pressure freezing and freeze substitution instruments for the electron microscopy (EM) Core facility at the University of Chicago. This technology will allow biologists to perform advanced EM by taking advantage of recent developments in immunocytochemistry and electron tomography. Compared to standard aldehyde fixation, cryofixation has the advantage of immobilizing cellular structures within milliseconds, thereby avoiding common fixation artifacts such as shape changes and membrane fusion. Cryofixation is useful for immunocytochemistry because structure can be well preserved without loss of antigenicity. Performing cryofixation at high pressures allows relatively thick samples to be frozen without being damaged by ice crystal formaton. This method is particularly appropriate for electron tomography, which enables biological structures to be reconstructed in three dimensions at nanometer resolution. In recent years, electron tomography has become a powerful tool for studying cellular processes such as organelle remodeling, membrane trafficking, and mitotic spindle dynamics. Several researchers at the University of Chicago have collaborated with groups at other institutions to perform high-pressure freezing and/or electron tomography of yeast, protist, and mammalian cells, and other cell biology and physiology researchers here need these techniques to answer pressing scientific questions. Users of the new techniques will include postdocs, graduate students, and undergraduates in University labs, as well as undergraduates who visit the University for summer research internships. High-pressure freezing has not been available in Chicago or the immediate vicinity, so the requested system will also be a valuable addition for researchers at nearby institutions. In addition, the new equipment and methods will be incorporated into classroom teaching at the graduate and undergraduate levels.

Testosterone and cortisol are some of the most intensively studied biological substances, yet our understanding of their behavioral effects in human beings is surprisingly limited. In many species, variations in hormone levels are powerful predictors of a wide variety of behaviors, and human beings should be no exception. The proposed research is designed to test a critically important idea underlying hormones and behavior -- that individuals high in testosterone possess a need or desire to maintain high status.
The core of this proposal seeks to test the hypothesis that differential testosterone levels, whether chronic or the result of transient changes, bear a functional relationship to status-seeking behavior. The initial experimental proposition put forth in this application is straightforward. After a competitive interaction, individuals either lose status or gain status. Then, they are given the option to attempt to regain their lost status or defend their high status. The primary prediction associated with this experiment is that individual differences in testosterone will predict a person's intention to pursue further competition. Change in cortisol level, a reliable measure of the stress response, is predicted to influence behavioral intention as well, but only among individuals with high levels of testosterone, who presumably possess strong status-seeking motives. An implicit measure of unconscious status seeking and power, the Picture Story Exercise will also be administered as a means of validating the hormonal measures. Further confirmation and validation of these measures will be assessed through cognitive activation of status needs using a modification of the Stroop color-naming procedure.
This research also has the potential to move experimental social psychology and the study of biological basis of social behavior forward through the innovative synthesis of these areas. Furthermore, this work will provide the theoretical foundation for future research that addresses the chronic problem of representation, retention, and achievement of talented individuals from underrepresented groups who may have status concerns. This enhanced understanding could inform strategies to increase the participation of women and minorities in science, engineering, and math.

For centuries, societies have wrestled with the question of how to balance the rights of the individual versus the greater good (for example, protection of civil rights versus protection from terrorist attacks). Many currently pressing debates in our society and urgent challenges have such issues at their root. To act (or omit acting) in order to protect the individual rights of one person, independent of any consideration of the broader context or broader consequences, abides by the moral principle of deontology. On the other hand when the well-being of a large number of people will be harmed as a result of those decisions it creates a dilemma, as this violates a moral principle of utilitarianism. Because both moral principles are intuitively plausible, they can lead to psychological conflicts within individuals and ideological conflicts in society when the two principles suggest different conclusions in a particular situation. To provide a basis for the resolution of such conflicts, the proposed research will examine the psychological processes underlying people's responses to moral dilemmas, their situational and individual determinants, and their effects on moral behavior in different contexts.

To address the study aims, the project will test and utilize a newly developed mathematical model that quantifies the unique contributions of three components that determine people's responses to moral dilemmas. Two are deontology and utilitarianism; the third is people's general tendencies toward action and inaction. In a series of 14 studies, the researcher will examine the contribution of cognitive and emotional processes to moral dilemma responses, the role of neuroendocrine factors in shaping moral decisions, and downstream effects of these processes on morally relevant behavior such as cheating and helping. By offering fine-grained measurements of the components, the proposed studies will provide deeper insights into the psychological processes underlying moral judgments and valuable practical implications for the resolution of moral controversies in society. In addition to illuminating the causes and consequences of moral judgments, the findings of this project will contribute to the scientific understanding of human action by identifying the conditions under which moral thoughts and feelings do and do not result in corresponding moral actions. The project also makes a valuable contribution to the development of research infrastructure by offering a novel tool for detailed analyses of moral judgments that will be freely available to other researchers.