A role for Tctex-1 (DYNLT1) in controlling primary cilium length

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Abstract

The microtubule motor complex cytoplasmic dynein is known to be involved in multiple processes including endomembrane organization and trafficking, mitosis, and microtubule organization. The majority of studies of cytoplasmic dynein have focused on the form of the motor that is built around the dynein-1 heavy chain. A second isoform, dynein heavy chain-2, and its specifically associated light intermediate chain, LIC3 (D2LIC), are known to be involved in the formation and function of primary cilia. We have used RNAi in human epithelial cells to define the cytoplasmic dynein subunits that function with dynein heavy chain 2 in primary cilia. We identify the dynein light chain Tctex-1 as a key modulator of cilia length control; depletion of Tctex-1 results in longer cilia as defined by both acetylated tubulin labeling of the axoneme and Rab8a labeling of the cilia membrane. Suppression of dynein heavy chain-2 causes concomitant loss of Tctex-1 and this correlates with an increase in cilia length. Compared to individual depletions, double siRNA depletion of DHC2 and Tctex-1 causes an even greater increase in cilia length. Our data show that Tctex-1 is a key regulator of cilia length and most likely functions as part of dynein-2.

Introduction

Primary cilia are found on nearly all cells in the human body (Ishikawa and Marshall, 2011, Satir et al., 2010). They are a major mechanosensory organelle with key roles in developmental patterning and cell growth control. Dysfunction of primary cilia is associated with a growing number of diseases including polycystic kidney disease and a large array of ciliopathies (Baker and Beales, 2009). The core of the primary cilium is formed by the axoneme, a 9 + 0 array of microtubules. These microtubules are marked by acetylation allowing specific detection using an antibody to detect this post-translational modification (Piperno and Fuller, 1985). The small GTPase Rab8a has been shown to be required for the formation and function of cilia (Nachury et al., 2007, Yoshimura et al., 2007) and can be used as a marker of the cilia membrane (Hattula et al., 2006) which while contiguous with the rest of the plasma membrane forms a functionally distinct domain. Trafficking with the cilium occurs by intraflagellar transport (Scholey, 2008), a process by which particles are translocated along the axoneme driven by kinesin-2 in the anterograde direction and dynein-2 in the retrograde direction. The particles that are moved are responsible for delivery of components necessary to build and maintain the cilium as well as to remove components and balance the growth of the axoneme and membrane.

The microtubule motor cytoplasmic dynein (Paschal et al., 1987, Schroer et al., 1989) has clear roles in microtubule organization, mitosis, organelle structure and positioning, and membrane trafficking (Vallee et al., 2004). In vertebrates, the dynein motor is built around a heavy chain subunit that provides ATPase-dependent force generation as well as microtubule coupling. Associated with this large subunit are a number of accessory subunits that appear to provide some functional specialization of the motor (King et al., 2002). There are two isoforms of the cytoplasmic dynein heavy chain in humans (Gibbons et al., 1994, Vaisberg et al., 1996), DHC1 (DYNC1H1) is the best studied and can be considered the canonical dynein motor responsible for the core functions of dynein. DHC2 (DYNC2H1) (Gibbons et al., 1994) is associated with unique isoforms of other dynein subunits: an intermediate chain (FAP133 in Chlamydomonas reinhardtii, DYCI-1 in Caenorhabditis elegans, and WDR32 in humans (Ishikawa and Marshall, 2011, Rompolas et al., 2007)), a light intermediate chain LIC3 (DYNC2LI1, also called D2LIC) (Grissom et al., 2002, Mikami et al., 2002, Perrone et al., 2003), and light chain LC8 (DYNLL1). Note that throughout we use the common names for the dynein subunits with the gene name as defined by Pfister et al. (2005) in the first instance of each case. Expression of DHC2 and LIC3 is consistent with a cilia function (Mikami et al., 2002) and compelling evidence exists for a role of these two subunits in the formation and function of primary cilia (Pazour et al., 1998, Pazour et al., 1999, Porter et al., 1999, Signor et al., 1999). Dynein-2 is a principle motor for retrograde intraflagellar transport within the cilium (Scholey, 2008). Notably, cells lacking D2LIC also lack monocilia (Rana et al., 2004) and show defects in embryogenesis. Mutations in DHC2 cause asphyxiating thoracic dystrophy and short rib-polydactyly syndrome, type III (Dagoneau et al., 2009) which are likely also ascribable to defects in cilia. Intriguingly though, in Tetrahymena thermophila dynein-2 regulates cilia length but is not itself required for ciliogenesis in Rajagopalan et al. (2009).

It is intriguing that so little is known of the role for the other subunits of cytoplasmic dynein in the function of dynein-2 or indeed in the process of ciliogenesis or intraflagellar transport. The light chain LC8 has been shown to be involved in this latter process in Chlamydomonas reinhardtii (Pazour et al., 1998) and indeed this work provided early compelling evidence of a role for dynein as the principle retrograde motor for intraflagellar transport.

In previous work we have used RNA interference (RNAi) to define the roles of individual subunits of the cytoplasmic dynein motor in intracellular membrane trafficking (Palmer et al., 2009). While previous work has shown that dynein-2 localizes to the Golgi (Grissom et al., 2002), we were unable to define any role for dynein-2 in ER-to-Golgi transport, Golgi organization, recycling endosome function or lysosome distribution (Palmer et al., 2009). During this previous study we used an in vitro assay for the formation of primary cilia (serum starvation of human retinal pigment epithelial (RPE1) cells and acetylated tubulin labeling of primary cilia) to validate the efficacy of our DHC2 and LIC3 suppression (cited in Palmer et al., 2009 as unpublished observations). We also used our RNAi approach to determine whether the suppression of other dynein subunits had any effect on primary cilia. Using this approach we found that the suppression of the dynein light chain Tctex-1 caused consistent defects in cilia function, manifest by a dramatic increase in cilia length. This phenotype was indistinguishable from that seen on suppression of DHC2 and indeed further analysis showed that suppression of DHC2 using specific siRNAs caused a concomitant loss of Tctex-1 from cells consistent with a physical interaction. Thus, our data define Tctex-1 as a key regulator of cilia length and implicate it as a component of the dynein-2 motor.

Section snippets

Results and discussion

Our previous work included validation of siRNA duplexes against all known cytoplasmic dynein subunits (Palmer et al., 2009). The majority of this previous work was undertaken in HeLa cells and validated in RPE1. This latter cell line generates primary cilia on serum starvation and consequently we used this to test the requirement for physiological levels of expression of the other dynein subunits in ciliogenesis. Fig. 1 shows our validation of the efficacy of dynein-2 suppression in these

Materials and methods

All reagents were purchased from Sigma–Aldrich (Poole, UK) unless stated otherwise. GFP-Rab8a (Hattula et al., 2006) was a kind gift from Johan Peränen (Helsinki, Finland) and was transfected 24 h prior to imaging or fixation. Antibody sources were as follows: anti-alpha tubulin (Clone DM1A) was from Sigma–Aldrich, anti-acetylated tubulin (Sigma–Aldrich, Poole, UK; clone 6-11B-1 (Piperno and Fuller, 1985)), anti-giantin (rabbit polyclonal, Covance, Princeton, New Jersey), DHC2 and LIC3

Acknowledgments

We are very grateful to Jon Lane and members of the Stephens lab for helpful discussions and critical input into the manuscript. We are especially grateful to Richard McIntosh, Stephen King, Richard Vallee, Johan Peränen, and Viki Allan for reagents and to the reviewers for their insight and suggestions. This work was funded by a Non-Clinical Senior Research Fellowship (to DJS) and the University of Bristol.

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