Organism reproduction, development, and survival are dependent on the equal and accurate segregation of genetic material into two daughter cells. The cell ensures the fate of DNA by properly assembling a macromolecular structure called the mitotic spindle to align and segregate chromosomes. The spindle is composed of microtubules (MTs) and many associated proteins that regulate MT dynamics to organize and shape the spindle. There are many proposed models for how the spindle assembles, however, the contributions of centrosomes, kinetochores, and chromatin to this process are not clearly defined. Although centrosomes are a primary source of MT nucleation in somatic cells, chromatin-mediated MT nucleation also occurs. Chromatin-mediated spindle assembly is mediated by the RanGTP and Chromosome Passenger Complex (CPC) gradients in which downstream proteins of these gradients regulate MT dynamics and are important for spindle organization. Many cell cycle related therapeutic techniques are used to alter MT dynamics or motor proteins that are involved in spindle assembly. Therefore, understanding how the spindle is initially organized will be beneficial in characterizing the activity of MT associated proteins that are the targets of therapeutic agents. In this present proposal I will: 1) Determine how the spindle is organized in the absence of chromatin and kinetochores in which I will determine how MTs are nucleated and organized in spindles formed in the absence of these components in two model systems. 2) Define the mechanisms utilized by the RanGTP and CPC gradients for spindle assembly to test the hypothesis that in a normal cell these two gradients overlap and cooperate for proper spindle formation. I will test this model using a FRET-based sensor of the CPC to detect the distribution of the gradient when the RanGTP gradient has been suppressed. Together these experiments will allow for further understanding of how these MT nucleating factors coordinate for proper spindle organization.

Public Health Relevance

Equal segregation of DNA into two daughter cells is essential for organism survival and development. This process is carried out by the mitotic spindle, which is the macromolecular machine composed of microtubules and associated proteins that act to attach chromosomes and segregate them during mitosis. Many therapeutic agents are used to target components of the spindle, therefore understanding how the spindle is assembled will be important for development of biomedical treatments.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31GM099309-03
Application #
8531289
Study Section
Special Emphasis Panel (ZRG1-F05-A (20))
Program Officer
Gaillard, Shawn R
Project Start
2011-09-01
Project End
2014-07-31
Budget Start
2013-09-01
Budget End
2014-07-31
Support Year
3
Fiscal Year
2013
Total Cost
$25,958
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Biochemistry
Type
Schools of Medicine
DUNS #
603007902
City
Indianapolis
State
IN
Country
United States
Zip Code
46202