Collaborative Research: RUI:The role of Polo Kinases in maintaining chiral asymmetry in the ciliate, Tetrahymena thermophila.

This project investigates how ‘simple’ single-cell protozoa express organizational complexity rivaling that seen in higher organisms. Protozoa are rarely in the public mind, except as potential pathogens. What’s overlooked is that protozoa have all the functions of larger organisms, but confined to a single cell. The ciliate Tetrahymena exhibits particular virtuosity by organizing surface structures into exquisitely complex geometries. Animals have mastered the art of organizing their tissues along two major axes, one defining anterior/posterior (heads vs tails), the other defining dorsal/ventral (back vs front). In contrast, ciliates have expanded their organizational geometry beyond this to develop circumferential patterning as well, so that their tiny cilia (used for swimming) are symmetrically arranged in a circular pattern around the cell, while more complex organelles are distributed at precise, and unique cellular longitudes. This project investigates how genes shared with humans are deployed by ciliates to achieve this 360-degree circumferential pattern. As such, it opens an entirely new cellular landscape for scientific exploration. This easily-grown organism also provides an attractive model for training undergraduates, as both labs continue to produce next-generation scientists via both summer research, and undergraduate laboratory courses involving advanced microscopy. Ciliates provide a novel paradigm for studying intracellular pattern in living cells. Animals organize cell and tissue types primarily along two dimensions, the anterior-posterior and dorsal-ventral axes, with modest differentiation between left and right. Individual animal cells are typically polarized in one dimension, along the apical-basal axis. One must look to some of the ‘simplest’ life forms (the ciliated protozoa) to discover mechanisms that go beyond two-dimensional cell planning, and manifest patterning in a 3rd dimension, that of circumferential patterning. Recent work by the two PIs has identified a suite of genes encoding Polo kinases and PKA homologs, that localize to hundreds of basal bodies populating the ciliate cell cortex. These kinases decorate basal bodies in discrete cortical domains, and may play a role in licensing specific meridians along which organelles assemble (e.g. the oral apparatus and contractile vacuole pores). The PIs hypothesize that ciliate pattern is driven by phosphorylation cascades radiating from basal bodies that serve as cortical signalosomes. In this model, cortical phosphorylation domains interact, to repress or reinforce one another across the cell surface licensing regions of the cortex to initiate construction of organelle assembly platforms. This study offers access to a genuinely novel intracellular patterning landscape. This investigation will utilize targeted gene knockouts, GFP-tagging, and morphological manipulation to interrogate cortical pattern mechanisms in the ciliate Tetrahymena thermophila. Localization and co-localization of gene products in wild-type and mutant cells as well as within cells undergoing pattern reorganization following cortical disruption will be explored with the aim of understanding how various kinases control organelle assembly along specific cortical meridians. This project is funded by the Cellular Dynamics and Function program of the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.