Kim S. McKim - US grants

9723330 McKim To investigate the mechanism of chromosome pairing in D. melanogaster, a combined approach of genetics, molecular biology and cell biology will be applied. Several aspects of the problem of initiating meiotic chromosome pairing and recombination will be addressed. Very little is known about the gene products that are required for this process. Two genes with important roles in this process, mei-W68 and mei-P22, have recently been identified. The protein product of these genes will be identified and the expression pattern of the transcript and protein will be characterized. One of the ways to understand this process is to characterize mutants defective in meiotic recombination. The temporal expression pattern of these genes will indicate when they are required. Only one gene involved in this process has been cloned and its function remains unknown. Both genes will be characterized. The object of this proposal is to investigate the mechanism of meiotic homolog pairing and recombination in Drosophila melanogaster females. During meiosis, paired homologous chromosomes are held together by a structure known as the synaptonemal complex (s.c.). Despite the ubiquitous nature of the s.c., there is considerable controversy on how it forms, its components, and its relationship to meiotic recombination. The studies in this proposal will enhance the understanding of two crucial issues in meiotic chromosome behavior. First, how do meiotic chromosomes pair and synapse; how much is based on DNA sequence similarity, and how much is based on "pairing sites". Second, what gene products have a role in this process, and what is the function of those gene products.

A confocal microscope system will be housed in the Waksman Institute at Rutgers University as a shared resource for ten different laboratories. A confocal microscope is needed in many studies to optically section through thick specimens with high resolution and sensitivity, and to simultaneously detect the signals from multiple targets, each labeled with a different fluorescent dye. The entire system includes a compound microscope equipped for fluorescence and DIC optics, a laser scanning and confocal detection system, a computer and software to operate the instrument and analyze the data collected, and a vibration-free table and workbench for the instrument. This system will replace an older confocal microscope, which has become obsolete and is no longer supported by its original manufacturer. The technological improvements of the new confocal microscope system are numerous and will allow many kinds of experiments that could not be performed previously to be done.

This instrument will be used for fluorescence microscopy of animal (Drosophila, C. elegans), fungal (S. cerevisiae) and plant (Arabidopsis, Tobacco) tissues. These studies address a wide range of scientific questions in developmental and cell biology. These include investigations of signal transduction pathways, regulation of tissue growth, regulation of gene expression, meiotic recombination, mechanisms that regulate the subcellular localization of different proteins, and synapse formation. These studies primarily involve the localization of proteins using fluorescently-tagged antibodies or other dyes. In addition, intrinsically fluorescent proteins, such as the Green Fluorescent Protein, are also employed. A confocal microscope that can optically capture sections through thick sections is absolutely essential for these experiments. In addition, numerous experiments will specifically take advantage of the many advanced capabilities of the new confocal microscopes, especially the higher resolution and sensitivity that will allow smaller and fainter signals to be observed. Other important improvements multiple lasers and detectors that provide the possibility to simultaneously observe multiple signals.

The users are addressing a range of scientific questions of critical importance in cell and developmental biology, and this instrument will enable them to make major contributions to their respective fields. These studies will provide many new insights into basic cellular processes that occur in plants and animals. In addition, the instrument will be made available to qualified users from outside of the Institute, and will play an important role in keeping Rutgers at the leading edge of studies in cell and developmental biology.

Meiosis is geared towards ensuring the segregation of homologous chromosomes at the reductional division by forming links between the homologs. The homologs are brought together in an elaborate pairing process that may begin with chromosomes aligned at a distance. This process culminates in a more intimate association where the homologs are held together along their entire length by the synaptonemal complex (sc). During this time meiotic recombination occurs, probably initiated with a double strand break (DSB). The repair and resolution of the repair process is believed to occur within a sc-associated, the recombination nodule (RN). Through our analysis of meiosis in Drosophila melanogaster females it has been shown that a key component of machinery that initiates meiotic recombination is conserved. In contrast, it has also been shown that the relationship between meiotic recombination and sc formation is not conserved. Long-term goals are to understand the mechanism and regulation of meiotic recombination and homolog pairing. Genetic and cytological techniques will be used to test the model that sc forms before recombination is initiated and genetic and chromosomal factors that regulate DSB formation will be identified and characterized. Specific aims are: To test the model that recombination initiates after sc formation. To identify genetic factors regulating double strand break formation. To identify chromosomal factors regulating double strand break formation.

Knowledge of the mechanisms resulting in chromosome pairing is critical to an understanding of inheritance. The results of this study will result in greater insight into phenomena central to a complete understanding of genetic mechanisms utilized by eukaryotic cells.

[unreadable] DESCRIPTION (provided by applicant): During the first meiotic division, homologous chromosomes linked by chiasmata interact with the spindle microtubules and segregate to opposite poles. Defects in this process lead to aneuploidy in the fertilized egg and have serious consequences on development, often resulting in death of the developing embryo. In humans, aneuploidy is a leading cause of spontaneous abortions and infertility in women. If aneuploids do survive, they manifest with diseases such as Down's, Tumer's or Klinefelter's syndrome. In many organisms, including humans and flies, the female meiotic spindle lacks centrioles and the classical microtubule-organizing center at the poles. The centrosomes and their constituent proteins are usually thought to organize bipolar spindles and therefore, in female meiosis, spindle pole organization must occur by a novel mechanism. By utilizing the powerful genetic and cytological techniques available in studies involving Drosophila melanogaster females, it is our long-term goal to elucidate the mechanism by which acentrosomal meiotic spindles form and how they function to segregate the chromosomes. We have found that the subito (sub) gene is required for bipolar spindle formation and homolog segregation during female meiosis in Drosophila. The findings that sub encodes a kinesin-like motor protein and is required during early embryogenesis and male meiosis has stimulated several new experiments. The aims of this study are to identify the role of SUB in spindle formation and understand how the meiotic spindle forms and functions to segregate chromosomes in the absence of centrosomes. A combination of genetic, immunological and cytological techniques will be used to illuminate the functions of SUB. These studies will investigate three critical areas of spindle biology by: 1) determining the subcellular localization of the sub protein in meiotic spindles and characterize how SUB interacts with the microtubules, 2) determining what other proteins interacts with SUB, and 3) using live imaging techniques to investigate the mechanism of spindle pole formation through a real time analysis of meiosis and mitosis in wild-type and mutants. The results of these studies will provide insights into the mechanisms of spindle formation and homolog segregation during meiosis and mitosis.

In most organisms, a programmed double strand break (DSB) initiates a repair process that results in either a crossover or a noncrossover. Crossovers function to direct segregation of homologous chromosome pairs at the first meiotic division. The principal investigator's long-term goal is to understand the mechanism that determines whether DSB repair in Drosophila female meiosis results in a crossover or noncrossover. Previous studies in Drosophila females suggest that there is one set of genes responsible for selecting crossover sites (precondition) and a second set required for the reaction to generate crossovers (exchange). In exchange mutants the investigator has detected delays in the timing of certain events during meiotic prophase. In a phenomenon similar to that observed in other organisms, errors in synapsis or recombination may trigger a delay in the progression of meiotic prophase. In this project, the principal investigator's group will test the hypothesis that, in Drosophila females, there is a surveillance mechanism specific for the crossover DSB repair pathway. The laboratory will characterize strains with mutations in genes known to function in conserved checkpoint pathways and determine if the mutations suppress the delays in prophase progression observed in exchange mutants. This will involve the genetic and cytological characterization of strains with single mutations and those with various combinations of two mutations. Antibodies recognizing synaptonemal complex components C(3)G and C(2)M, a protein required for DSB formation (MEI-P22), and a histone modification at DSB sites will be used for the cytological studies. Interestingly, preliminary evidence suggests that the step in the crossover pathway which triggers the delay does not depend on the presence of DSBs. Thus, the investigator will be particularly interested in a surveillance mechanism that may detect errors in homolog pairing or chromosome structure that are independent of DSBs. These experiments should provide new insights into meiotic surveillance mechanisms. Indeed, understanding more about a surveillance mechanism for crossover formation and knowing what is monitored should provide clues as to how crossover sites are established.

This laboratory puts a significant effort into providing research training to undergraduate students. Twenty-seven undergraduate students have spent at least one research semester (and usually at least one year) in the principal investigator's laboratory since the spring of 1997. Senior Genetics undergraduates spend over 20 hours per week working on their individual thesis projects. High school students have also spent summers performing research in the laboratory. This project will also benefit the University community, as its cytological experiments require the use of a confocal microscope (purchased with the support of an NSF Instrumentation grant), and the members of the McKim lab are responsible for the maintenance of this microscope, for training and assisting users from other laboratories, and for updating the instrumentation and techniques. Finally, the laboratory frequently distributes Drosophila stocks and research resources to the scientific community, often prior to publication.

DESCRIPTION (provided by applicant): During the first meiotic division, homologous chromosomes linked by chiasmata interact with the spindle microtubules and segregate to opposite poles. Defects in this process lead to aneuploidy in the fertilized egg and have serious consequences on development, often resulting in death of the developing embryo. In humans, aneuploidy is a leading cause of spontaneous abortions and infertility in women. If aneuploids do survive, they manifest with diseases such as Down's, Turner's or Klinefelter's syndrome. In many organisms, including humans and flies, the oocyte meiotic spindle lacks centrioles and the classical microtubule-organizing center at the poles. Since the centrosomes and their constituent proteins usually organize bipolar spindles, spindle pole organization in oocyte meiosis must occur by another mechanism. Drosophila melanogaster oocytes are an excellent system to elucidate the mechanisms of acentrosomal spindle formation. Subito is the Drosophila homolog of human Mitotic Kinesin Like Protein 2 (MKLP2) and is required for bipolar spindle formation during female meiosis. Subito is required for the development of the central spindle at meiotic metaphase and this structure may be critical for formation of a bipolar spindle in the absence of centrosomes. The goals of this study are: i) analyze the structure and function of Subito. This will provide insights into how this protein functions and is regulated. ii) characterize the interactions between Subito and the Passenger proteins or the Ran pathway. This will determine which proteins interact with Subito and what is their function in spindle assembly. iii) determine the timing of bipolar spindle formation and chromosomes orientation. This will test the relative importance of interpolar and kinetochore microtubules and investigate how pairs of homologous chromosomes orient on the acentrosomal spindle. iv) analyze new genes required for meiotic spindle assembly. This will identify new genes required for acentrosomal spindle assembly using a relatively unbiased approach. During the first meiotic division, the chromosome number is reduced in half by separating pairs of homologous chromosomes into the gametes. In humans, errors in meiosis lead to aneuploidy, an abnormal number of chromosomes in a sperm or egg, and is the leading cause of spontaneous abortions, infertility in women and diseases such as Down's, Turner's or Klinefelter's syndrome. The objective of these studies is to understand how these errors occur.

DESCRIPTION (provided by applicant): During the first meiotic division, homologous chromosomes linked by chiasmata interact with spindle microtubules and segregate to opposite poles. Defects in this process lead to aneuploidy in the fertilized egg and usually in death of the developing embryo. In humans, aneuploidy is a leading cause of spontaneous abortions and infertility in women and causes diseases such as Down, Turner or Klinefelter syndromes. In many organisms, including mammals and insects, the oocyte meiotic spindle lacks centrosomes. In the absence of the microtubule-organizing center found at mitotic spindle poles, the chromosomes generate a signal which stimulates spindle assembly. In cells with centrosomes, the microtubule connections formed between the poles and the kinetochores facilitates bi-orientation of sisters (mitosis) or homologous chromosomes (meiosis I). In acentrosomal cells, novel mechanisms may be employed to bi-orient the homologs. We have found that a group of central spindle proteins, including the Chromosome Passenger Complex (CPC) is critical for formation of a bipolar spindle and orientation of the homologs. These proteins recruit and organize the antiparallel microtubules overlap in the center of the spindle. How these microtubules mediate chromosome behavior is not known. In this proposal, we will investigate the mechanisms of homolog orientation in the acentrosomal spindle of Drosophila oocytes. The CPC is recruited to chromosomes even in the absence of microtubules but unlike mitotic cells, CPC proteins are not found at the centromeres. Instead, the CPC is found in a ring around the chromosomes where it recruits factors which regulate spindle assembly such as Subito. The ring structure also provides a mechanism for directing spindle bipolarity in the absence of centrosomes. To investigate the role of the central spindle in homolog orientation, we will use fluorescently tagged proteins and live imaging to investigate the timing of homolog orientation relative to spindle assembly and establishment of the central spindle. We will also determine the role of kinetochores in chromosome alignment and segregation. Since these genes are essential, we will use sophisticated genetic tools available in Drosophila to generate oocytes lacking these proteins. This includes newly developed germ line RNAi and germ line clones to test the role of different kinetochore components. Finally, we will test the hypothesis that the bi-orientation of homologs depends on an interaction between chromosome associated and central spindle microtubules.

During the first meiotic division, homologous chromosomes linked by chiasmata attach to microtubules from opposite spindle poles (bi-orientation) and then segregate. In humans, errors in chromosome segregation in the oocyte lead to aneuploidy and are the leading cause of miscarriage, infertility and birth defects. Our long-term goal is to understand the mechanisms that promote accurate chromosome segregation, and the features of the oocyte spindle that make it susceptible to chromosome segregation errors. Our previous research using Drosophila melanogaster females has led to a model in which two types of microtubule attachment are used for bi-orientation. Lateral attachments, where the kinetochores interact with the sides of microtubules, establish bi-orientation. Then end-on attachments, where the kinetochores attach to the end of microtubules, maintain and segregate bi-orientated homologs. A prominent feature of the Drosophila oocyte is the metaphase I central spindle, which functions as a ?backbone?, organizing the microtubules into a bipolar structure in the absence of centrosomes. Our work has shown that the central spindle has an important role in bi-orientation during pro-metaphase. Studies in C. elegans and mouse oocytes indicate that the metaphase central spindle may be a conserved element required for the bi-orientation of homologous chromosomes during acentrosomal meiosis. In the previous funding period, we developed several tools to study the mechanisms of bi- orientation in oocytes. These tools include RNAi resistant transgenes in order to make germline-specific mutants of key proteins. Furthermore, we have the reagents, either transgenes or antibodies, to detect many of the important proteins that regulate chromosome segregation, including centromere, kinetochore, checkpoint and spindle proteins. With these tools, we will investigate the mechanisms by which the central spindle interacts with the kinetochores to promote bi-orientation. It is likely that premature stabilization of end-on attachments leads to bi-orientation defects. Therefore, we will investigate the mechanisms of lateral attachments and bi-orientation, and how the transition to end-on attachments is regulated. These studies will focus on two kinetochore proteins, CENP-C and SPC105R, which are required to load several other kinetochore and checkpoint proteins. We will also investigate how the central spindle interacts with the kinetochores and promotes accurate bi-orientation. These studies will include experiments to model in Drosophila, central spindle mutations that decrease fertility in human females. The Aims of this proposal are linked by a goal to understand the mechanisms of chromosome segregation important to oocytes. In completing this work, we will have gained insights into how kinetochores regulate the transition from lateral and end-on microtubule attachment.