The long-term objective of this work is to understand the molecular basis of regulated mitotic spindle orientation. Alignment of the mitotic spindle along a predetermined axis is required for proper cell function in many contexts, including differentiation, embryogenesis, and organogenesis. For example, during the asymmetric division of Drosophila neuroblasts, precursors of the central nervous system, cell fate determinants localize to opposite poles of the cell such that they become segregated into discrete daughter cells. Proper distribution of determinants, and subsequent fate specification, requires that the mitotic spindle align precisely with the polarity axis. We propose to investigate this process by reconstituting spindle orientation in a cultured cell that does not normally orient the spindle. Establishing regulated spindle positioning in this context will allow us to determine which components are sufficient for spindle orientation, and we propose to examine these components biochemically and structurally to determine their mechanism of action. In our preliminary work we have successfully polarized Drosophila S2 cells using the adhesion protein Echinoid and have found that expression of Echinoid proteins in which the cytoplasmic portion has been replaced with domains from the Partner of Inscuteable (Pins) protein can robustly orient the spindle in a manner similar to neuroblasts. We are using a combination of biochemical, biophysical, and cell biological methods to investigate the function of molecules that we identify in our spindle orientation reconstitution, including Pins.

Public Health Relevance

During cell division, chromosomes are separated into the daughter cells by the mitotic spindle. In many cells, the spindle must be precisely positioned for proper tissue organization, differentiation, or prevention of tumor formation. In this work, we are attempting to identify the cellular machinery required for spindle position control by attempting to recreate this process in a cell type that does not normally orient its spindle. As the loss of accurate spindle positioning is associated with human disease, improving our understanding of the molecules that control this process will contribute to our knowledge of the mechanisms of disease states.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM087457-04
Application #
8542865
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Deatherage, James F
Project Start
2010-09-10
Project End
2014-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
4
Fiscal Year
2013
Total Cost
$249,335
Indirect Cost
$67,818
Name
University of Oregon
Department
Biochemistry
Type
Organized Research Units
DUNS #
948117312
City
Eugene
State
OR
Country
United States
Zip Code
97403
Lu, Michelle S; Prehoda, Kenneth E (2013) A NudE/14-3-3 pathway coordinates dynein and the kinesin Khc73 to position the mitotic spindle. Dev Cell 26:369-80
Johnston, Christopher A; Manning, Laurina; Lu, Michelle S et al. (2013) Formin-mediated actin polymerization cooperates with Mushroom body defect (Mud)-Dynein during Frizzled-Dishevelled spindle orientation. J Cell Sci 126:4436-44
Johnston, Christopher A; Doe, Chris Q; Prehoda, Kenneth E (2012) Structure of an enzyme-derived phosphoprotein recognition domain. PLoS One 7:e36014
Mauser, Jonathon F; Prehoda, Kenneth E (2012) Inscuteable regulates the Pins-Mud spindle orientation pathway. PLoS One 7:e29611
Johnston, Christopher A; Whitney, Dustin S; Volkman, Brian F et al. (2011) Conversion of the enzyme guanylate kinase into a mitotic-spindle orienting protein by a single mutation that inhibits GMP-induced closing. Proc Natl Acad Sci U S A 108:E973-8
Smith, Nicholas R; Prehoda, Kenneth E (2011) Robust spindle alignment in Drosophila neuroblasts by ultrasensitive activation of pins. Mol Cell 43:540-9