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Mechanical basis for motor protein coordination in axenomes leading to the beating of cilia and flagella

Final Report Summary - DYNEIN COORDINATION (Mechanical basis for motor protein coordination in axenomes leading to the beating of cilia and flagella)

Introduction

Motile cilia play two essential roles in human health. First, they drive fluids across cell surfaces. This role is critical to clearing mucus in the respiratory system, moving the ovum down the Fallopian tube in the reproductive system, circulating cerebrospinal fluid in the brain, and establishing left-right polarity in development. Second, they propel single cells, such as spermatozoa, through fluids. Thus, when cilial defects occur, patients exhibit a serious consequences.

Healthy motile cilia oscillate with regular, repeatable waveforms. The waveforms are generated by the coordinated activity of tens of thousands of dynein motor proteins applying force to the microtubule structural scaffold within cilia. Coordinated activity must be regulated spatially (both around and along the cilia) and temporally; dynein on one side bend the cilia in one direction, and dynein on the opposite side bend it the other way. Several mechanisms are thought to influence coordination, including chemical and mechanical regulation pathways through the dynein. However, the regulation of dynein through its interaction with tubulin is not understood. We asked, what are the mechanism by which the mechanochemical properties of ciliary dynein are regulated by the microtubules?

Methods:

To answer this question, we used a single celled alga, Chlamydomonas reinhardtii, as a model organism. Chlamydomonas has two motile cilia that are structurally similar to all eukaryotic cilia because it is a highly conserved structure. We used and perfected protocols to culture, harvest, and purify dynein motor proteins from Chlamydomonas1. Then, we reconstituted the motility of dynein from cilia using in vitro motility assays1. These assays enabled the isolation of dynein-microtubule interactions, separate from other chemical and mechanical effects. Specifically, we assembled the purified dynein molecules on microscope slide and added microtubules and ATP, which is the energy source for the dynein motor proteins1. We recorded movies of the microtubules as they glided across the surface using fluorescent microscopy. We tracked the position of the microtubules in each frame using FIESTA2 and analyzed the data using custom analyses1.

Results:

This first thing we found was that, unlike most other motor proteins, dynein from cilia moves the microtubules with unsteady velocity in in vitro motility assays. Our analysis indicated that this unsteadiness is unlikely to be a result of artifacts introduced by the experimental conditions. However, we were concerned that it might be due to a mismatch between the source of dynein (Chlamydomonas) and the source of microtubules (pig), which is a condition mitigated in more steady studies by using a mammalian source of motor proteins. This mismatch occurred in our assays because Chlamydomonas tubulin, the building blocks from which microtubules are polymerized, is impossible to purify with traditional techniques. Therefore, we developed a novel tubulin purification protocol, and applied it to tubulin from Chlamydomonas cilia3. We compared the motility of Chlamydomonas dynein on Chlamydomonas and pig microtubules. We found that they both exhibited unsteadiness4.
Because the unsteadiness was present in dynein motility on both Chlamydomonas and pig microtubules, we developed an analysis technique, called displacement-weighted velocity analysis, to quantify the unsteadiness in a systematic and unbiased way4. We found that not only is unsteadiness present, but also it is quantitatively unchanged with the switch to Chlamydomonas from pig microtubules4.
Despite there being no difference in the unsteadiness, we found that the dynein can recognize the difference between the microtubules using displacement-weighted velocity analysis. By analyzing the dependance of microtubule gliding motility on microtubule length, we found that dynein moves faster on short Chlamydomonas microtubules than on shorter pig microtubules4. Using a model for the gliding assay that bridges molecular and cell length scales, we found that this difference suggests that the dynein has a higher affinity for Chlamydomonas microtubules than for pig microtubules4.
Next, we investigated the mechanism by which dynein recognizes the microtubule. Porcine and Chlamydomonas tubulin are different. They share only ~85% amino acid sequence identity, and they are post-translationally modified (PTM) differently. It has been suggested that PTMs can affect motor motility5, though not confirmed for ciliary motors. Using motility assays in which we altered the lysine 40 acetylation state of the microtubules biochemically in vitro, we found that dynein recognizes the differences between the modified and unmodified tubulins6. In this case, the effect is that the speed of long microtubules is higher for acetylated microtubules than for those who have not been acetylated6. Using the model for gliding assays, we found that dynein's powerstroke, the state of the mechanochemical cycle in which the force is transmitted to the microtubule, gets faster with increasing amount of microtubule acetylation6.
Additionally, a series of PTMs occur in a region of the tubulin called the E-hook5. By treating microtubules with a protease, we cleaved the E-hooks off the microtubules, and, by comparing the motility of dynein on treated and untreated microtubules, we found that motility is higher on microtubules without E-hooks6. Again, by modeling the result, we found that dynein adjusts its inherent speed in response to E-hooks. This suggests that E-hook associated PTMs such as detyrosination, polyglutamylation, and polyglycylation5 can affect the motility of dynein.

Conclusions and outlook:

We discovered that the motility of the dynein, the motor that powers the beat of cilia, can be regulated by the chemistry of tubulin making up the microtubule structure of the cilium. These results suggests that, along with chemical and mechanical regulation pathways through dynein, cells can regulate ciliary beating by controlling ciliary microtubule chemistry. This may explain why particular PTMs and tubulin isoforms are known to locate to the cilia7. These results also shed further light on the problem of the tubulin code, particularly on the role of tubulin regulation in mitosis, microtubule dynamics, and intracellular transport8. They also have implications for motor protein regulation and show that motility should be thought of as a protein-protein interaction with the microtubule track. Additionally, the techniques we developed through this work, including the novel tubulin purification protocol and the displacement-weighted velocity analysis technique, will have transformative impacts in the study of tubulin and unsteady motile processes, respectively.

We are currently following up with several aspects of this work. We expect that these results, in conjunction with the results of this study and studies by other research labs around the world, will give a more compete understanding of dynein coordination mechanisms in cilia. They will contribute to a bottom-up multi-scale model of the ciliary beat connecting the single molecule properties of dynein to the cell and tissue level phenomena. This level of understanding could lead to both treatments of ciliary disease and engineering of biological nanostructures.

References:

1. Alper, J., Geyer, V., Mukundan, V. & Howard, J. in Methods Enzymol. (Marshall, W. F.) Volume 524, 343–369 (Academic Press, 2013).

2. Ruhnow, F., Zwicker, D. & Diez, S. Tracking Single Particles and Elongated Filaments with Nanometer Precision. Biophys. J. 100, 2820–2828 (2011).

3. Widlund, P. O. et al. One-step purification of assembly-competent tubulin from diverse eukaryotic sources. Mol. Biol. Cell 23, 4393–4401 (2012).

4. Alper, J. D., Tovar, M. & Howard, J. Displacement-Weighted Velocity Analysis of Gliding Assays Reveals that Chlamydomonas Axonemal Dynein Preferentially Moves Conspecific Microtubules. Biophys. J. 104, 1989–1998 (2013).

5. Janke, C. & Bulinski, J. C. Post-translational regulation of the microtubule cytoskeleton: mechanisms and functions. Nat Rev Mol Cell Biol 12, 773–786 (2011).

6. Alper, J. D., Decker, F., Agana, B. & Howard, J. Tubulin modification directly affects axonemal dynein motility. Prep. (2013).

7. Ludueña, R. F. Multiple forms of tubulin: different gene products and covalent modifications. Int. Rev. Cytol. 178, 207–275 (1998).

8. Verhey, K. J. & Gaertig, J. The Tubulin Code. Cell Cycle 6, 2152–2160