The PI of this Mentored Career Development Award proposal has significant experience in biophysics theory, and a long history of working closely in collaboration with experimentalists. However, her lack of direct experimental training has proved to be a severe limitation in her progress toward her long-term career objective: to advance the understanding of chromosome motions and mitosis, and related human-health issues, through an integrated experimental and theoretical approach. This grant will provide protected training time in order for the PI to: (1) Learn to design and conduct ex- periments. This will require practical laboratory training in a range of techniques in biophysics, cell biology, molecular biology, genetics, and biochemistry. (2) Educate herself in biology to complement her physics training and to allow her to formulate cutting-edge, biologically relevant research questions. This will require participation in conferences, workshops, seminars, and journal clubs. (3) Develop her expertise as she manages an experi- mental laboratory. This will require training in lab safety, grantwriting, and the responsible conduct of research. The research component of the project will address the capture of kinetochores (KCs) by microtubules (MTs) in cell division. For years the primary mechanism of KC capture in mitosis was believed to be microtubule search and capture, in which dynamic MTs grow in different directions from centrosomes and make end-on attachments with KCs. However, recent work found that lateral KC attachment to rotationally diffusing MTs enabled rapid KC capture even with significantly reduced MT dynamics. Previous work has focused exclusively on MT dynamic instability or rotational diffusion and therefore has been unable to compare the two mechanisms and determine their relative importance.
The specific aims are: 1: Evaluate the importance of microtubule dynamic instability and rotational diffusion to kinetochore capture using quantitative imaging and a first-generation model. The preliminary model developed for this proposal is believed to be the first model of KC capture that includes both MT dynamic instability and MT and KC diffusion. Measurements will be made of the fraction of lost KCs and polar MT lengths as a function of time after recovery from cold block will determine the time course of KC capture;the data will be used to fit unknown parameters in the model. The model will allow assessment the importance of MT rotation and dynamic instability, separately and together, for KC capture. 2: Measure the dynamics of mitotic nuclear polar microtubules and use the measured parameters to create a second-generation kinetochore capture model. The study in Aim 1 requires fitting key model parameters for MT dynamic instability. Because uncertainty in model parameters leads to uncertainty in model predictions, for a reliable and accurate model it is best to use measured values of MT dynamic instability pa- rameters, rather than relying on estimates or fits. However, dynamics of nuclear polar MTs in mitosis have not previously been measured in sufficient detail to build a quantitative model without unknown parameters. Because these MTs are short-lived, several live-cell imaging approaches will be compared to determine MT dynamics parameters. 3: Predict and measure how alterations in microtubule dynamics affect the time course of kinetochore capture. Biological perturbations such as disease states can lead to alterations in MT dynamics, but it is not understood how these alterations affect KC capture. Current models are not able to predict how MT dynamics affect KC. The model will be used to find """"""""sensitive"""""""" regions of parameter space where small changes in MT dynamics parameters lead to relatively large changes in the time course of KC capture. Multiple experimental perturbations are available to perturb MT dynamics in fission yeast. The results will test the understanding of the contributions of MT dynamic instability and rotational diffusion to KC capture. The PI will be mentored under this grant by J. Richard McIntosh, an esteemed scientist whose work has focused on cell division in the model organism S. pombe. He will provide lab space and equipment to allow the PI to fulfill the aims of this grant. This will be an ideal training environment, as McIntosh has recently retired from teaching, though still maintains an active research lab. The PI is an associate professor at the University of Colorado, Boulder. There are several initiatives in place at CU Boulder that have fostered a strong interdisciplinary research environment. The PI is part of the Biofrontiers Institute, and attends monthly meetings with her biophysics colleagues from a range of departments. The institution is committed to fully supporting the PI as she learns techniques in cell biology.
Capture of chromosomes by the mitotic spindle is thought to be important for preventing aneuploidy, the mis-segregation of chromosomes that is associated with cancer and birth defects. This project will study the role of microtubule polymerization dynamics and rotational diffusion in chromosome capture in fission yeast, a model organism for the study of cell division.
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