LASER MICROBEAM IRRADIATION: KINETOCHORE & CHROMOSOMES IN CRANE FLY SPERMATOCYTE

激光微束照射：动粒

• 批准号: 6280701
• 负责人: JAMES LAFOUNTIAN
• 金额: $ 1.63万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6280701
• 关键词: Invertebrata; biomedical resource; biotechnology; growth /development; lasers; microscopy; reproductive system; technology /technique

This project involves laser microbeam surgery on meiotic chromosomes in crane fly spermatocytes. The laser is being used to create acentric chromosome fragments and/or to destroy kinetochores during different stages of meiosis. The effects of these procedures on the movement of the chromosomes is then monitored with the video DIC. Results are expected to reveal the transport properties of the meiotic spindle complex and provide evidence for movement of chromosomes or chromosome fragments that lack kinetochores. First, we saw that in cold induced laggards, the telomere fragment of the chromosome that normally is oriented toward the pole and migrates towards the equator in meiosis, instead leads to the pole when severed. Secondly, during anaphase, the acentric fragments that usually trail behind the centrosome, instead move towards the equator when severed by the laser. During the June 2-June 4 visit of Dr. LaFountain we saw that telomere-containing fragments of trailing chromosome arms during anaphase moved after they were severed from the centromere-containing half-bivalents. During normal anaphase, such telomere-containing fragments moved toward the equator and beyond into the opposite half spindle. Telomere-containing fragments from cold-recovering laggards moved in the other direction--toward the pole. During the June 11-12 visit, we used cold-recovering cells to show that movement of fragments -- both types from normally segregating half-bivalents and those from cold-recovering laggards -- could occur in the same half spindle. Fragments from laggards moved toward the pole as fragments from normally segregating chromosomes moved toward the equator and beyond. That experiment showed that fragments always move in the direction with their telomeres leading. A second experiment was done to try to localize the domain of a fragment that is essential for movement. Theoretically, a moving fragment could have motors all along its surface, or it could have the motors for movement localized to the telomeric tip (the portion that always leads the way during movement after severing from the original chromosome). For this, we released telomere-containing fragments from normally segregating half-bivalents. During transit to the opposite pole, we then made a second cut to create a smaller telomere-containing fragment and an interstitial fragment lacking a telomere. In all cases, the interstitial fragment stopped moving after the second cut, but the telomere-containing fragment continued toward the opposite pole. That result implicates the telomere in the mechanism of fragment movement. Whether the telomere contains molecular motors that power fragment movement, or whether the telomere is tethered to its partner at the opposite pole, we cannot discriminate between these possibilities at present. Experiments aimed at resolving these issues may be possible in future visits. Lastly, we tried to release telomere-containing fragments from chromosomes during the second division of meiosis using secondary spermatocytes. We did so in one cell, the other attempts were not successful. the results suggested that cutting of chromosomes during the second division must be done early is anaphase. We apparently cut too late in anaphase, after the mechanism for movement was no longer active. The July 1-2 sessions were primarily to gather data on the rates of acentric fragment movement after they had been released during anaphase. The general result on rates is that fragments initially move quite rapidly (6 (m/min) after being severed from the chromosome, but their velocities diminish to about 0.5 - 1.0 (m/min as they move beyond the equator and into the opposing half-spindle. Also during the July 1-2 visit, I attempted to remove kinetochores from bivalents at metaphase and then to record their movement subsequent to release. The goal of that was to create a very small fragment containing the 2 kinetochores of one of the homologues and a very large fragment containing the two kinetochores of the other homologue plus the large acentric portions of the homologue from which the small fragment was removed. i am still in the process of analyzing the results of those experiments, but the impression gained so far is that the kinetochores (plus the very small amount of chroma tin attached) move toward the pole much faster than the other large mass that contains the bulk of the chromatin and its two attached kinetochores. If further analysis bears out that difference, then the movement of the two fragments created by that operation may be load dependent, which is not in agreement with what the literature has to say about chromosome movements. I will need to get more data on this point, but at this juncture, it looks very interesting. LaFountain, J.R., R.W. Cole and C.L. Rieder. (1997) Laser microsurgery of anaphase chromosomes in crane-fly spermatocytes: Kinetochore-independent movement of telomere-containing chromosome fragments. Mole. Biol. Cell 8: (in press)