C&SP30: Modeling of Tetrahymena basal body dynamics

A. Collaborating Investigators: Chad G. Pearson, ¹ Robert F. Murphy²

B. Institutions: ¹University of Colorado and ²Carnegie Mellon University

C. Funding Status of Project: 5R01GM099820-04 Mechanisms of Centriole Assembly and Stability (Pearson) 9/24/12-7/31/17

 D. Biomedical Research Problem:

Mapping basal body assembly in Tetrahymena cells. The Pearson laboratory works on understanding how basal bodies assemble through the cell cycle in Tetrahymena cells. While most of this is directed at specific molecules in the assembly process, the Pearson lab is also interested in mapping where, spatially within Tetrahymena cells, basal bodies assemble and to understand where they go / separate upon assembly. This will allow us to explore the pattern that basal bodies organize into for normal ciliary beating and hydrodynamic flow. Therefore, the goals of this collaboration are the following:

 

1. Analyze the data previously acquired by the Pearson lab ¹⁰⁹ to map the stereotypical organization of basal bodies relative to a) the cell cycle and b) the cell size. This will then be the basis for future work to model in ciliary beating and the efficiency of cellular motility. Importantly, because larger cells during cell division swim more slowly, we predict that the slower motility reflects changes in basal body positioning. We will also use these analyses to establish a baseline for future mutant analyses.

2. Modeling localized basal body separation. This is a micro-version of aim 1 and will establish the temporal dynamics of new, daughter basal body separation from old, mother basal body separation. Such studies are currently ongoing in the lab to assess protein incorporation into basal bodies as new basal bodes separate from their mother basal body and mature. We currently make the assumption (likely incorrect) that as basal bodies separate from their mother that this is a linear proxy for their age. We will use CellOrganizer¹¹⁰ to define the temporal separation of basal bodies. These studies will also be correlated with ongoing studies following the dynamics of protein incorporation at newly forming basal bodies.

3. Live imaging of basal body separation. In a direct test of aim 2, we will visualize “parts of” basal body separation during assembly and maturation and use CellOrganizer¹¹⁰ (TR&D4) to reassemble our “parts” to the full view. This will reveal the rate of basal body separation relative to basal body protein incorporation and structural maturation.

Mapping the Tetrahymena ciliary beat pattern. We want to measure / reconstruct the ciliary beat pattern in Tetrahymena cells. Using DIC imaging and high temporal resolution imaging (~500fps), we have low resolution imaging of the Tetrahymena ciliary beat pattern. We will marry these data with fixed timepoint fluorescence data to reconstruct a generative model of the Tetrahymena cilia, basal body and associated structures during the ciliary beat stroke.

 

E. Methods and Procedures. There are two basic methods in CellOrganizer that will be used for this project. The first is to automatically assemble collections of static images of fixed cells that are at various points in the cell cycle into a model of the changes in basal body arrangement through the cycle. This will be done by measuring pairwise distances between images and assembling them into a shape space as we have done previously for cell and nuclear shape analysis.⁸⁵ The second is to take short movies of living cells and assemble them into a dynamic shape space as has been done previously for the dynamics of H1299 cell shape changes. The main difference is that the distance measure will be based on basal body positions rather than overall cell shape.