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      Dynamics of the IFT machinery at the ciliary tip

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          Abstract

          Intraflagellar transport (IFT) is essential for the elongation and maintenance of eukaryotic cilia and flagella. Due to the traffic jam of multiple trains at the ciliary tip, how IFT trains are remodeled in these turnaround zones cannot be determined by conventional imaging. Using PhotoGate, we visualized the full range of movement of single IFT trains and motors in Chlamydomonas flagella. Anterograde trains split apart and IFT complexes mix with each other at the tip to assemble retrograde trains. Dynein-1b is carried to the tip by kinesin-II as inactive cargo on anterograde trains. Unlike dynein-1b, kinesin-II detaches from IFT trains at the tip and diffuses in flagella. As the flagellum grows longer, diffusion delays return of kinesin-II to the basal body, depleting kinesin-II available for anterograde transport. Our results suggest that dissociation of kinesin-II from IFT trains serves as a negative feedback mechanism that facilitates flagellar length control in Chlamydomonas.

          eLife digest

          Cilia and flagella are hair-like structures that protrude from nearly every human cell and play a number of roles including transmitting signals and enabling cells to move. These structures lengthen when new material is deposited at their tip by a process called intraflagellar transport (IFT). In this process, protein complexes known as IFT trains carry cargo along tracks that run along the length of each flagellum. Different motor proteins power the IFT trains in different directions: kinesin moves IFT trains from the base to the tip, while dynein moves them back in the opposite direction.

          When IFT trains arrive at the tip of the flagellum, they release their cargo and undergo a major reorganization process in which the trains switch motors in order to move back to the base. Because the many IFT trains at the tip form a kind of ‘traffic jam’, standard imaging techniques have been unable to distinguish exactly what happens during this reorganization.

          A new imaging method called PhotoGate microscopy can track individual molecules inside crowded cells. Chien, Shih et al. have now used this method to visualize the full range of movements made by IFT trains and motors in the flagella of a species of single-celled algae. This revealed that at the tip of the flagellum, IFT trains split apart and mix with each other to assemble into new trains, which move back to the base. In addition, kinesin carries dynein to the tip as inactive cargo, detaches from IFT trains at the tip and diffuses back to the base of the flagellum. This delays kinesin’s return and causes it to accumulate in the flagellum, which helps to explain why flagella assemble more slowly as they lengthen: as the flagellum grows longer, less kinesin is available at the base to transport material to the tip. Thus kinesin diffusion helps the algae to regulate the length of their flagella.

          Further research is now needed to study whether similar mechanisms control the length of flagella in other organisms. Defects in the intraflagellar transport process have been linked to a range of human diseases known as ciliopathies, and so the results presented by Chien, Shih et al. could also help us to uncover the causes and potential treatments for these conditions.

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          Most cited references 47

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          The depolymerizing kinesin MCAK uses lattice diffusion to rapidly target microtubule ends.

          The microtubule cytoskeleton is a dynamic structure in which the lengths of the microtubules are tightly regulated. One regulatory mechanism is the depolymerization of microtubules by motor proteins in the kinesin-13 family. These proteins are crucial for the control of microtubule length in cell division, neuronal development and interphase microtubule dynamics. The mechanism by which kinesin-13 proteins depolymerize microtubules is poorly understood. A central question is how these proteins target to microtubule ends at rates exceeding those of standard enzyme-substrate kinetics. To address this question we developed a single-molecule microscopy assay for MCAK, the founding member of the kinesin-13 family. Here we show that MCAK moves along the microtubule lattice in a one-dimensional (1D) random walk. MCAK-microtubule interactions were transient: the average MCAK molecule diffused for 0.83 s with a diffusion coefficient of 0.38 microm2 s(-1). Although the catalytic depolymerization by MCAK requires the hydrolysis of ATP, we found that the diffusion did not. The transient transition from three-dimensional diffusion to 1D diffusion corresponds to a "reduction in dimensionality" that has been proposed as the search strategy by which DNA enzymes find specific binding sites. We show that MCAK uses this strategy to target to both microtubule ends more rapidly than direct binding from solution.
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            The primary cilium at a glance.

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              Small-molecule inhibitors of the AAA+ ATPase motor cytoplasmic dynein

              The conversion of chemical energy into mechanical force by AAA+ (ATPases associated with diverse cellular activities) ATPases is integral to cellular processes, including DNA replication, protein unfolding, cargo transport, and membrane fusion 1 . The AAA+ ATPase motor cytoplasmic dynein regulates ciliary trafficking 2 , mitotic spindle formation 3 , and organelle transport 4 , and dissecting its precise functions has been challenging due to its rapid timescale of action and the lack of cell-permeable, chemical modulators. Here we describe the discovery of ciliobrevins, the first specific small-molecule antagonists of cytoplasmic dynein. Ciliobrevins perturb protein trafficking within the primary cilium, leading to their malformation and Hedgehog signaling blockade. Ciliobrevins also prevent spindle pole focusing, kinetochore-microtubule attachment, melanosome aggregation, and peroxisome motility in cultured cells. We further demonstrate the ability of ciliobrevins to block dynein-dependent microtubule gliding and ATPase activity in vitro. Ciliobrevins therefore will be useful reagents for studying cellular processes that require this microtubule motor and may guide the development of additional AAA+ ATPase superfamily inhibitors.
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                Author and article information

                Contributors
                Role: Reviewing Editor
                Journal
                eLife
                Elife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                20 September 2017
                2017
                : 6
                Affiliations
                [1 ]deptBiophysics Graduate Group University of California, Berkeley BerkeleyUnited States
                [2 ]deptPhysics Department University of California, Berkeley BerkeleyUnited States
                [3 ]deptDepartment of Genetics, Cell Biology and Development University of Minnesota MinneapolisUnited States
                [4 ]deptDepartment of Molecular and Cell Biology University of California, Berkeley BerkeleyUnited States
                Utrecht University Netherlands
                Utrecht University Netherlands
                Author notes
                [†]

                These authors contributed equally to this work.

                Article
                28606
                10.7554/eLife.28606
                5662288
                28930071
                © 2017, Chien et al

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                Product
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100000057, National Institute of General Medical Sciences;
                Award ID: GM055667
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000057, National Institute of General Medical Sciences;
                Award ID: GM094522
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000001, National Science Foundation;
                Award ID: MCB-1055017
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000001, National Science Foundation;
                Award ID: MCB-1617028
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000057, National Institute of General Medical Sciences;
                Award ID: GM116204
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Categories
                Research Article
                Biophysics and Structural Biology
                Cell Biology
                Custom metadata
                During intraflagellar transport (IFT), kinesin-II dissociates from IFT trains at the flagellar tip and its diffusion in flagella serves as a negative feedback mechanism that facilitates flagellar length control in Chlamydomonas.

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