Greenfield Sluder - US grants

Centrosomes form the poles of the spindle in animal cells and are involved in microtubule nucleation, chromosome alignment, chromosome movement, and determining the plane of the cleavage furrow. The ways in which the centrosomes are formed, function, and reproduce are poorly understood. 1. We have isolated centrioles from sea urchin sperm and have microinjected them into fertilized sea urchin eggs. This always leads to the formation of numerous supernumerary asters. To test for the role of the putitive centriolar RNA in centrosome formation and reproduction, we will treat the centrioles with various RNAases, a RNA crosslinking compound, or irradiate them with 260nm light in the presence of ethidium bromide. 2. We will study the role of centrosomal RNA in microtubule nucleation in vitro. Isolated centrosomes will be chilled or treated with Colcemid to disassemble existing microtubules. They will then be exposed to 6S tubulin before or after treatment by RNAases, fragments of RNAases, RNA crosslinking and RNA breaking compounds. We will determine the ability of the treated isolates to nucleate microtubules in vitro. 3. Isolated centrosomes will be treated in the ways just described and then microinjected into fertilized eggs. We will test for microtubule nucleation, chromosome attachment, chromosome movement, and reproduction of the centrosomes. 4. We will investigate the functional properties of the pericentriolar material when separated from centrioles in vivo. Portions of the pericentriolar material will be pulled off the centrosome with a microneedle and their behavior followed with a polarizing microscope. We will look for the ability of this pericentrolar material to function as a pole and reproduce with each cell cycle. These experiments should help resolve the controversy over the role of the centriole in the formation of the spindle pole. 5. We will investigate how the poles of acentriolar, anastral spindles are established. We will microinject plant or mouse egg spindles into fertilized eggs. Their behavior in the sea urchin cytoplasm will be determined. This work will provide a better understanding of the mechanisms that control cell division in both normal and cancerous cells.

This proposal is a request for funds to help support the 1987 Northeast Regional Conference on Developmental Biology. The Conference will be held at the Marine Biological Laboratory, Woods Hole, Massachusetts, September 24-27, 1987. The goal of the Conference is to bring together developmental biologists from the Northeast to present their work in formal sessions and informal discussions. A special effort is being made to bring together investigators at all career stages: graduate students to established investigators. The conference will include 5-6 platform sessions, a poster session and a keynote address. The purpose of the conference is to provide an effective opportunity for the formal and informal exchange of information and ideas between established investigators and graduate or postdoctoral students. This goal is achieved much more readily at the regional conferences than at national meetings primarily because the regional conferences are smaller, less rigidly scheduled, and allow ample time and occasion for discussions.

Centrosomes are the spindle poles in dividing cells and are necessary for microtubule nucleation, chromosome alignment, chromosome movement, and establishment of the plane of the cleavage furrow. The ways in which the centrosomes form, function, and reproduce are poorly understood. We want to learn how these processes are controlled in order to establish a basis for new technologies to control cancer cell proliferation. 1. We isolate centrioles from sea urchin sperm and microinject them into fertilized sea urchin eggs. This leads to the formation of numerous supernumerary asters. To test for the role of the putative centriolar RNA in centrosome formation and reproduction, we will treat the centrioles with various RNAases, and psoralen, a RNA crosslinking compound. 2. We will inject nucleases or psoralen treated centrioles from sea urchin sperm and basal bodies from Chlamydomonas into activated Xenopus eggs to determine if asters from during meiosis or interphase. This should resolve the discrepancy between our existing results and those of another group. 3. To define the similarities and dissimilarities between acentriolar-anastral spindle poles and typical animal centrosomes, we will transfer the anastral- acentriolar spindles from mouse eggs, a mutant Drosophila cell line, and plant cells into fertilized sea urchin eggs. We want to examine the activity of these spindles in the sea urchin cytoplasm. Later we will characterize their ultrastructure to determine if they have undergone any structural changes after one or more cell cycles in the sea urchin egg. 4. We will determine the relative roles of centrioles and pericentriolar material in the reproduction of centrosomes. We will treat eggs with acridine orange and use a focused laser microbeam to ablate centrioles or portions of the pericentiolar material. By serial section ultrastructural analysis, we will correlate the structures damaged with the reproductive capacity of the centrosome. 5. We will determine if centrosome formation and reproduction in sea urchin eggs is under translational control. 6. Several exploratory projects are briefly described.

In broad terms our research is directed at control mechanisms for cell division: both the events of mitosis and the cell cycle regulation for entry into and exit from mitosis. This proposal describes a number of studies centered on the interrelationship between centrosomes and the cell cycle with an emphasis on checkpoint control mechanisms. Using echinoderm zygotes, frog egg extracts, and cultured cells as model systems, we will: 1. Test the existence of a proposed checkpoint for entry into mitosis that monitors centrosome/centriole duplication. 2. Investigate how centrosome reproduction is coordinated with the nuclear cell cycle. 3. Investigate how centriole duplication is controlled during the cell cycle. 4. Continue our characterization of how the checkpoint for the metaphase/anaphase transition functions in sea urchin zygotes. 5. Continue our studies of the control of nuclear envelope breakdown and the checkpoint for the completion of DNA synthesis. 6. Investigate how the reproductive capacity of maternal centrosomes is controlled in starfish eggs. By characterizing the functional properties of control mechanisms in living cells, we can address issues that are not readily amenable to biochemical analysis. The health relevance of this work is that an essential characteristic of malignant cells is abnormal regulation of the proliferative cycle. Thus, a better understanding of how the mitosis portion of the cell cycle is controlled is necessary for the development of practical strategies to control cancer. Indeed, a number of currently used chemotherapeutic drugs exploit defects that transformed cells have in the checkpoint mechanisms that control entry into mitosis and control exit from mitosis at the metaphase/anaphase transition.

In broad terms our research is directed at elucidating the control mechanisms for cell division. We use living cells and egg extracts to provide information that cannot be obtained from conventional biochemical and molecular approaches. 1. We developed the first Xenopus egg extract that supports multiple rounds of centrosome reproduction. We will finish our characterization of this experimental system and then use it to further investigate the mechanisms that control centrosome reproduction and coordinate it with nuclear events in the cell cycle, with an emphasis on the role of cyclin dependent kinase 2-cyclin E (Cdk2-E) in the regulation of centrosome reproduction. 2. We will characterize the activities of Cdks complexed with cyclins A and E when sea urchin zygotes are arrested in G1 and S phases and correlate these activities with the different abilities of centrosomes to repeatedly reproduce in these cell cycle phases. 3. We will use mammalian somatic cells to test whether the initiation of centriole duplication can occur during G1, without cell cycle progression into S phase. This should provide new insight into the centriole cycle and its cell cycle regulation. 4. We will test the hypothesis that the spindle, not the cytoplasm, provides the competency for the cell to execute the metaphase-anaphase transition. 5. We will use vertebrate somatic cells to study the regulation of chromatid cohesion at the metaphase-anaphase transition. Health relevance: We study the control of centrosome reproduction because a wide variety of human tumors have an abnormally high number of centrosomes; this can directly lead to aneuploidy and genomic instability through the formation of multipolar spindles at mitosis. We study the regulation of chromatid cohesion, because failure of the sister chromatid arms to completely release at anaphase onset directly leads to chromosome breakage or chromosome non-disjunction, either of which leads to genetic damage that can promote neoplastic transformation.

A group composed of eight NIH funded principal investigators requests funds to purchase a sensitive, high-resolution, multimode digital microscope system. All eight are tenured or tenure-track faculty at the UMASS Medical School. The entire group works on the same floor of a research building near the UMASS Medical School. There is a current and pressing need, associated with the NIH funded research of these laboratories, for video time lapse microscopy of living cells using a variety of imaging, analysis, and display modalities. There are no general use time lapse systems with suitable capabilities at the UMASS Medical Center. The proposed microscope system will be used for basic research in the fields of cancer cell biology, neurobiology, developmental biology, cell cycle regulation, control of gene expression, and cell motility. The proposed instrument system will allow these researchers to take their investigations in new directions, obtain novel information, and thus, significantly advance their programs in basic biomedical research.

DESCRIPTION (provided by applicant): The centrosome serves as the primary microtubule-organizing center of the cell and has a profound influence on all microtubule dependent processes. The interphase centrosome duplicates before mitosis and the daughter centrosomes define the two poles of the mitotic spindle. Our research is directed at elucidating the control mechanisms for cell division with an emphasis on learning more about the interrelationship between centrosomes and the cell cycle, particularly non-traditional activities of the centrosome. We analyze the functional properties of living cells to provide information that cannot be obtained by conventional means and that will set the stage for further biochemical or molecular studies. 1. We will complete and extend our work aimed at understanding how centrioles influence the ability of the cell to progress through GI. 2. Cells from which the centrosome has been laser ablated will reassemble multiple centrosomes during prolonged S phase. To test concerns that this is due to residual fragments of the original centrosome that "seed" the assembly of new centrosomes, we will use mechanical microsurgery to remove the interphase centrosome and determine if truly new centrosomes can reassemble. 3. When mammalian somatic cells fail to cleave, they arrest in G1 if they have a functional p53 pathway. Cleavage failure is a critically important event to monitor because it leads to extra centrosomes and multipolar mitosis. This causes genomic instability that can contribute to the genesis of cancer. We will conduct a number of functional studies to determine how the cell knows that it is polyploid. 4. A key issue in centrosome biology is how human tumor cells develop the extra centrosomes that lead to genomic instability and genetic imbalances. Some hold that centrosome amplification is simply the consequence of cleavage failure while others claim that centrosomes can reduplicate during a single cell cycle. We will test if centrosomes can reduplicate within a cell cycle under conditions where cleavage failure is not a factor.

DESCRIPTION (provided by applicant): Centrosomes nucleate most spindle microtubules and thus, determine spindle polarity. Since the essential bipolarity of mitosis depends upon the cell containing just two centrosomes, centrosome duplication must be under tight numerical and temporal control. Cleavage failure and centrosome reduplication are thought to be the most probable causes of centrosome amplification (>2 centrosomes at mitosis). Extra centrosomes raise he chances for spindle multipolarity and consequent unequal chromosome distribution. This leads to the aneuploidy and genomic instability that drives the evolution of the transformed state. Our research uses individual living cells to investigate the controls for cell division with an emphasis on the interrelationship between centrosomes and the cell cycle. Aim 1: We will characterize the process of centriole/centrosome reduplication during prolonged S phase in living CHO and U2OS cells. We also will characterize human papillomavirus oncoprotein E7 induction of centrosome amplification. Aim 2: We will investigate the importance of centrosome localization sequence (CLS) dependent binding of cyclins A and E to the centrosome for the duplication and reduplication of the centrosome. Aim 3: For normal human cells we will characterize the functional consequences of cleavage failure in the genesis of persistent centrosome amplification and investigate the proliferative capacity of tetraploid cells. We will determine if cleavage failure can produce persistent centrosome amplification in CHO cells that have a mutated p53 and do not arrest when tetraploid. Aim 4: We will investigate the relationship between the duration of mitosis and the ability of daughter cells to progress through G1 in normal human cells. We will investigate why a modest (>1 hour) prolongation of mitosis causes the daughter cells of a seemingly normal division to arrest in G1. Aim 5: We will investigate whether centriole structure is determined by the self-assembly characteristics of its subunits or by a template present at the mother centriole. We will introduce C. elegans (worm) centrioles with 9 singlet microtubules into Xenopus (frog) egg extracts that support duplication of centrioles with 9 triplet microtubules. We will determine if worm centrioles are functional in a heterologous cytoplasm and examine the ultrastructure of the daughter centrioles to determine if they have the worm, the frog, or some other structure.

DESCRIPTION (provided by applicant): We will investigate the controls for centriole duplication and how prolonged prometaphase blocks daughter cell proliferation. Since mitotic fidelity depends upon the cell containing just two centrosomes, centrosome duplication must be under tight numerical and temporal control. Extra centrosomes (centrosome amplification) at mitosis can lead to unequal chromosome distribution and consequent genomic instability which is a driving force in multi- step carcinogenesis. In Aim 1 we test a model that can explain centrosome amplification after DNA damage, a well established but poorly understood phenomenon. Radiation and radiomimetic drugs are currently used to treat human tumors. Follow up radiation therapy can cause DNA damage in proliferating normal cells in the tumor region - particularly after surgery. In Aim 2 we will investigate if and how geminin plays a role in enforcing the block to centrosome reduplication during S and G2 phases of the cell cycle. The centrosome intrinsic block to reduplication is of critical importance in preventing centrosome amplification. In Aim 3 we test if targeting of Cdk2-cyclin E to the centrosome is required for centrosome duplication or if soluble pools of this kinase complex are sufficient to drive centrosome duplication. This explores the importance of local control of centrosome duplication and provides insight into how zygotes control centrosome duplication despite constitutively high cytoplasmic Cdk2-cyclin E activity. In Aim 4 we will further characterize the basis for our discovery that prolonged prometaphase causes an irreversible block to daughter cell proliferation despite the normal division of the mother cell. This proliferation block can serve to handle mitotic defects due to environmental toxins that are never resolved but allow satisfaction of the mitotic checkpoint and consequent improper completion of mitosis. We will also explore the possibility that chemotherapeutic regimes using microtubule targeting drugs could lead to stem cells withdrawing from the cell cycle thereby compromising tissue regeneration, wound healing, and tissue maintenance.