Tubulin Polymerization-Promoting Protein Family Member 3 (Tppp3) Facilitates Microtubule Bundling and Network Formation via its Weak Interaction with Microtubules

Abstract

Tubulin polymerization-promoting protein family member 3 (TPPP3) is a conserved protein factor found in cytoplasm associated with microtubules in many ciliated cells. The expression level of TPPP3 in mouse tracheal epithelial cells (mTEC) was reported to vary in concomitant with the formation of microtubule networks in the cytoplasm during epithelia development. These observations suggest that TPPP3 takes part in formation of the microtubule network. Here, we expressed several TPPP3 constructs and, by using conventional biochemical measurements and single molecule observations, examined its effects on cell morphism of HEK293 cells, the binding, the bundling, and the elongation of microtubules in vitro. TPPP3 formed microtubule networks in vitro in a concentration dependent manner and showed specific but weak binding (Kd = ca. 5 μM) to the microtubules. The molecular dissection combined with single molecule observations suggests that the ability to dimerize the TPPP3 molecules resides in the N-terminal domain and the ability to promote microtubule elongation resides in the C-terminal domain of TPPP3. Micro-rheology measurements of TPPP3-microtubule network were carried out, in which the Brownian motion of microspheres of 0.2 μm-diameter in the microtubule network was measured. The measurement showed that the network was crosslinked but remained flexible. This crosslinked flexibility enables the network to adopt the optimal mechanical configuration in response to the external force. These results suggest that during the development of the basal body array in ciliated cells, the loose but somehow restricted network provides the adaptability of the array to the external load. This work was supported by funding from the Dynamic Mechanisms and Fundamental Technology for Biological Systems and the Creation of Fundamental Technologies for Understanding and Control of Biosystem Dynamics, CREST, from Japan Science and Technology.