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Branched F-actin as a negative regulator of cilia formation
E XP E RI ME N T AL C ELL RE S EA RC H
319 (2013) 147–151
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/yexcr
Review Article
Branched F-actin as a negative regulator of cilia formation Xiumin Yan, Xueliang Zhun State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China
article information
abstract
Article Chronology:
Cilia dysfunction leads to developmental defects and also a spectrum of human diseases termed
Received 26 July 2012
ciliopathies. The actin cytoskeleton is a highly dynamic network and involved in many
Received in revised form
important biological processes, such as cell migration and membrane trafficking. Recently,
30 August 2012
actin dynamics has been shown to play a critical role in ciliogenesis. This review summarizes
Accepted 31 August 2012
these results and provides insight into possible mechanisms.
Available online 10 September 2012
& 2012 Elsevier Inc. All rights reserved.
Keywords: Primary cilia Actin Vesicle
Contents Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Primary cilium is a microtubule-based organelle that protrudes from the cell surface like an antenna. The base of primary cilium is a specialized centriole named basal body, whereas the ciliary axoneme contains nine pairs of microtubule doublets lying beneath the ciliary membrane. Primary cilium is widely distributed in cells of higher animals. It provides a specialized compartment for signal transduction of several signaling pathways including the hedgehog pathway [1–3]. Its dysfunctions have been shown to cause a number of human genetic diseases, such as polycystic kidney, polydactyly, blindness and situs inversus [1,2,4]. Primary cilia form in G0 or G1 phase of the cell cycle and are disassembled prior to M phase entry [2,5]. Serum deprivation, or ‘‘starvation’’, has thus been widely used to induce cilia formation
n
Corresponding author. Fax: þ86 21 5492 1011. E-mail address: [email protected] (X. Zhu).
0014-4827/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexcr.2012.08.009
in cultured cells. The initiation of ciliogenesis requires conversion of mother centriole into a basal body that serves as the ciliary foundation. The ciliary axoneme then elongates, with the aid of the bidirectional intraflagellar transport (IFT), to form a cilium. Proteins or other components critical for cilia structure and function are transported into cilia by microtubule-dependent molecular motors kinesin or dynein [1,2,6]. Therefore, disruption of either the formation of basal body or the IFT hinders cilia assembly [6,7]. Emerging lines of evidence have recently shown that actin dynamics contributes markedly to ciliogenesis and cilia length. The filamentous actin (F-actin) forms divergent and dynamic cytoskeleton networks in cells. Among the different forms of
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F-actin are branched F-actin and stress fibers [8]. Branched Factin is nucleated by the ARP2/3 complex and mainly distributed in the cell cortex. It forms lamellipodium, a thin layer of F-actin network that propels membrane protrusions at the leading edge of migrating cells. It is also found to associate with membrane vesicles and function in vesicle sorting and trafficking [8–10]. While serum starvation usually requires more than 12 h to induce robust ciliation [11], the treatment of cytochalasin D, an F-actin destabilizer, provokes rapid ciliogenesis in 1–2 h [12,13]. Moreover, cilia length increases by approximately 2-fold when cytochalasin D or B is applied to serum-starved cells [12–14]. More importantly, cytochalasin D exerts these cilia-promoting effects at low concentrations incapable of affecting the integrity of stress fibers [12], implying that cilia formation is promoted through the inhibition of certain highly dynamic form(s) of F-actin. Follow-up studies revealed that branched F-actin is inhibitory to cilia formation. In a high-throughput RNAi screen in telomereimmortalized human retinal pigmented epithelial (RPE1) cells, Kim and colleagues found that the depletion of ARP3 induces ciliogenesis in the presence of serum and an increase in cilia length in serum-starved cells [12]. ARP3 is a subunit of the ARP2/3 complex responsible for the nucleation of branched F-actin [9]. Consistently, we reported that overexpression of mir-129-3p, a microRNA, stimulates ciliation in cycling mammalian cells and also increases cilia length by concomitantly downregulating four positive regulators of branched F-actin, ABLIM1, ABLIM3, TOCA1 and ARP2 [13]. ARP2 and TOCA1 are subunit and activator of the ARP2/3 complex [9,15], respectively. ABLIM1 and ABLIM3 are F-actin-associated proteins [16,17] important for lamellipodia formation [13], though
319 (2013) 147–151
how the ABLIM proteins affect branched F-actin in lamellipodia is currently unclear. Furthermore, although our results suggest that mir-129-3p needs to target all four proteins to maximize its influence on ciliation, the ablation of any one of them is actually sufficient to phenocopy the effects of the mir-129-3p overexpression. The mir-129-3p overexpression and the cytochalasin D treatment do not display additive effects on either ciliogenic efficiency or cilia length, suggesting that they both affect similar cellular machineries [13]. Therefore, inhibition of branched F-actin formation is a common theme in these studies. These results also provide a reason to serum starvation-induced ciliogenesis because serum starvation is well-known to repress lamellipodia formation [18,19]. The inhibitory effect of the knockdown of Missing-in-Matastasis (MIM) on starvation-induced ciliogenesis [14] may also be attributed to branched F-actin. The ablation of MIM, a monomeric globular actin (G-actin)-binding protein [20], stimulates actin polymerization by enhancing the Src-dependent phosphorylation of Cortactin [14,21]. Cortactin is a class-II nucleation-promoting factor (NPF) of the ARP2/3 complex and also able to promote the formation of branched F-actin together with class-I NPFs such as the WASP family proteins WASP, N-WASP, and SCAR/WAVE [9]. Interestingly, although MIM and Cortactin have been shown to colocalize extensively at the cell cortex [22], a portion of MIM is located at the basal body and this localization appears to be important for its role in ciliogenesis, suggesting the importance of the regional inhibition of actin polymerization in cilia maintenance [14]. Although RNAi, overexpression and serum starvation are not physiological, the changes in actin dynamics are indeed of
Golgi Recycling endosome Plasma membrane protein Ciliary membrane protein Branched F-actin Vesicles Rab11 Rab8 BB (iv)
(ii)
(iii) Inhibition of branched F-actin
(i)
Fig. 1 – A model for roles of branched F-actin in ciliogenesis. (i) Rab8 vesicles are usually sorted from recycling endosomes with the help of branched F-actin and transported by microtubule-dependent motor kinesin (not depicted) to polarized membrane domains such as the lamellipodium. (ii) On the other hand, the accumulation of ciliogenic vesicles including those positive for Rab8, Rabll, and ciliary membrane proteins at the centrosome is critical for efficient ciliogenesis and cilia elongation. (iii) Under ciliogenic conditions, inhibition of branched F-actin impairs the regular vesicle sorting processes, resulting in generation and/or accumulation of ciliogenic vesicles at the centrosome region to promote cilia formation. (iv) The cortical actin network contributes to the cilia membrane domain (CMD) formation by tethering plasma membrane proteins and hence excluding them from the cilium.
EX P ER IM E NTA L CE LL R ES E A RC H
importance in ciliation in vivo. The antagonistic relationship between MIM and Cortactin appears to regulate cilia resorption before M entry because the levels of Cortactin phosphorylation and MIM alter inversely in G2/M phases of the cell cycle [14]. On the other hand, our studies suggest that mir-129-3p is a bona fida positive regulator of ciliation in vertebrate tissues. mir-129-3p is highly expressed in mouse tissues abundant in primary cilia, including brain, retina, kidney and testis. Its inhibition in zebrafish embryos suppresses ciliation in Kupffer’s vesicle and the pronephros, where mir-129-3p is highly expressed, and causes phenotypes typical of ciliapathies, including a curved body, pericardial edema, and defective left-right asymmetry [13]. mir-129-3p also contributes to the serum starvation-induced ciliogenesis because it is moderately upregulated in RPE1 cells after serum starvation and its neutralization inhibited ciliation [13]. Branched F-actin appears to impact ciliogenesis by modulating membrane trafficking. Electron microscopy revealed that a membrane vesicle associated with the distal end of the mother centriole is involved in the initial steps of ciliogenesis and forms subsequently the ciliary sheath [23–25]. Furthermore, cilium constitutes a special membrane domain into which membrane components and membrane proteins need to be delivered from the cytoplasm [3,6,25]. RAB8 is a small GTPase responsible for the vesicle trafficking to the polarized membrane domains such as lamellipodia of migrating cells and the apical side of epithelial cells, whereas RAB11 is important for membrane trafficking from the trans-Golgi network (TGN) and of the recycling endosome (Fig. 1) [26–28]. The ‘‘regular’’ functions of these RABs, however, can be switched to cilia-specific roles because RAB11 recruits and targets Rabin8, the guanine nucleotide exchange factor (GEF) for RAB8, to the centrosome to activate RAB8 for the IFT during ciliogenesis [29,30]. RAB8-positive vesicles becomes abundant at the centrosome area about 1 h after serum withdrawal [29]. Inhibition of branched F-actin by the ablation of ARP3, cytochalasin treatment, or overexpression of mir-129-3p has also been shown to promote the centrosomal accumulations of ciliogenic vesicles positive for Rab11 [12,13]. Therefore, inhibition of branched F-actin is important for the switch from ‘‘regular’’ to ciliogenic trafficking (Fig. 1). Detailed mechanism on how branched F-actin inhibits ciliogenic vesicle trafficking is still not known. Nevertheless, branched F-actin has recently been shown to widely participate in membrane sorting and trafficking by providing constricting forces in the form of a scaffold to facilitate membrane scission [10,31,32]. It is found to exist on different membrane organelles under the control of the WASP family NPFs such as WASH and WHAMM. Inhibition of its nucleation induces marked morphological changes, e.g. tubulation, in various membrane organelles and represses regular membrane trafficking [10,31–34]. RAB8 is enriched in vesicles at the leading edge. Cytochalasin D treatment enhances the formation of Rab8-positive membrane tubules, presumably by disrupting the membrane recycling route of endosomes [27,35]. Therefore, inhibition of branched F-actin possibly results in formation, re-distribution and trafficking of ciliogenic vesicles, including those positive for RAB8 and RAB11 (Fig. 1). In agreement with this postulation, PLA2G3, a phospholipase important for the generation and maintenance of membrane tubules [36–38], is found to serve as a positive regulator of ciliogenesis [12].
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149
Interestingly, it seems that not all branched F-actin is harmful to cilia because such a form of actin is also important for the selectivity of ciliary components. Periciliary diffusion barrier(s) are found to gate the entry into and exit from the cilia [39]. For instance, a size-exclusion permeability barrier has recently been found to regulate the ciliary concentrations of soluble proteins and other macromolecules using a mechanism analogous to that of the nuclear pore [40]. Furthermore, a disc-like ciliary membrane domain (CMD) excluding cortical F-actin and plasma membrane proteins such as podocalyxin and CEACAM1 also exists at the ciliary base in polarized MDCK cells [41,42]. Further studies indicate that the cortical actin network outside the CMD selectively tethers the membrane proteins depending on specific ‘‘retention signals’’ to exclude them from the CMD [41]. How the CMD lacks F-actin, however, is not clear. Could the basal body-localized MIM [14] hinder cortical actin polymerization there? Kim et al. [12] found in their RNAi screen that the ablation of two Gelsolin family F-actin severing proteins GSN and AVIL [43] leads to ciliogenesis defect, implying that during starvation-induced ciliogenesis certain F-actin-based structure(s) need to be chopped up. If the tethering mechanism is also effective in non-polarized cells, could the servering activity of GSN and AVIL be used to trim the cortical actin from the CMD? There are other unanswered questions on the relationship between cilia and the actin cytoskeleton as well. For instance, the fact that the inhibition of branched F-actin stimulates ciliogenesis also indicates a promotion of the mother centriole-to-basal body transition. The removal of CP110 from the distal end of the mother centriole is believed as an important step for the transition [5]. Although both serum starvation and the overexpression of mir129-3p downregulate CP110 [5,13,44], the cytochalasin D treatment fails to affect the global CP110 levels, nor do Cep97 and Kif24 [13] (data not shown), important for the centrosomal recruitment of CP110 [44,45]. Therefore, how the cytochalasin D treatment leads to the removal of CP110 from the mother centriole [13]remains elusive. Furthermore, inhibition of branched F-actin is not the only factor that impacts ciliogenesis. Serum starvation can generally induce ciliation in more than 80% of RPE1 cells, whereas cytochalasin D treatment or even the overexpression of miR-129-3p to 10000-fold over the endogenous level only stimulated ciliation in 50% of cells [12,13]. It is thus intriguing to elucidate how additional factor(s) synergize with actin dynamics to achieve maximal ciliogenic efficiency. Besides Rabin8, RPGR (retinitis pigmentosa GTPase regulator), a ciliopathy protein required for the ciliary localization of Rab8, also functions as a GEF for Rab8 during ciliogenesis [46]. Interestingly, knockdown of RAB8 or RPGR affects actin dynamics [27,47]. Therefore, it will be interesting to examine whether the RAB8-related changes in actin dynamics also has a role in cilia formation.
Acknowledgements
The authors are grateful to J. Cao for his help in preparing the illustration. This work was supported by the National Basic Research Program of China (2012CB945003 and 2010CB912102) and National Science Foundation of China (30971430).
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319 (2013) 147–151
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/yexcr
Review Article
Branched F-actin as a negative regulator of cilia formation Xiumin Yan, Xueliang Zhun State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China
article information
abstract
Article Chronology:
Cilia dysfunction leads to developmental defects and also a spectrum of human diseases termed
Received 26 July 2012
ciliopathies. The actin cytoskeleton is a highly dynamic network and involved in many
Received in revised form
important biological processes, such as cell migration and membrane trafficking. Recently,
30 August 2012
actin dynamics has been shown to play a critical role in ciliogenesis. This review summarizes
Accepted 31 August 2012
these results and provides insight into possible mechanisms.
Available online 10 September 2012
& 2012 Elsevier Inc. All rights reserved.
Keywords: Primary cilia Actin Vesicle
Contents Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Primary cilium is a microtubule-based organelle that protrudes from the cell surface like an antenna. The base of primary cilium is a specialized centriole named basal body, whereas the ciliary axoneme contains nine pairs of microtubule doublets lying beneath the ciliary membrane. Primary cilium is widely distributed in cells of higher animals. It provides a specialized compartment for signal transduction of several signaling pathways including the hedgehog pathway [1–3]. Its dysfunctions have been shown to cause a number of human genetic diseases, such as polycystic kidney, polydactyly, blindness and situs inversus [1,2,4]. Primary cilia form in G0 or G1 phase of the cell cycle and are disassembled prior to M phase entry [2,5]. Serum deprivation, or ‘‘starvation’’, has thus been widely used to induce cilia formation
n
Corresponding author. Fax: þ86 21 5492 1011. E-mail address: [email protected] (X. Zhu).
0014-4827/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexcr.2012.08.009
in cultured cells. The initiation of ciliogenesis requires conversion of mother centriole into a basal body that serves as the ciliary foundation. The ciliary axoneme then elongates, with the aid of the bidirectional intraflagellar transport (IFT), to form a cilium. Proteins or other components critical for cilia structure and function are transported into cilia by microtubule-dependent molecular motors kinesin or dynein [1,2,6]. Therefore, disruption of either the formation of basal body or the IFT hinders cilia assembly [6,7]. Emerging lines of evidence have recently shown that actin dynamics contributes markedly to ciliogenesis and cilia length. The filamentous actin (F-actin) forms divergent and dynamic cytoskeleton networks in cells. Among the different forms of
148
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F-actin are branched F-actin and stress fibers [8]. Branched Factin is nucleated by the ARP2/3 complex and mainly distributed in the cell cortex. It forms lamellipodium, a thin layer of F-actin network that propels membrane protrusions at the leading edge of migrating cells. It is also found to associate with membrane vesicles and function in vesicle sorting and trafficking [8–10]. While serum starvation usually requires more than 12 h to induce robust ciliation [11], the treatment of cytochalasin D, an F-actin destabilizer, provokes rapid ciliogenesis in 1–2 h [12,13]. Moreover, cilia length increases by approximately 2-fold when cytochalasin D or B is applied to serum-starved cells [12–14]. More importantly, cytochalasin D exerts these cilia-promoting effects at low concentrations incapable of affecting the integrity of stress fibers [12], implying that cilia formation is promoted through the inhibition of certain highly dynamic form(s) of F-actin. Follow-up studies revealed that branched F-actin is inhibitory to cilia formation. In a high-throughput RNAi screen in telomereimmortalized human retinal pigmented epithelial (RPE1) cells, Kim and colleagues found that the depletion of ARP3 induces ciliogenesis in the presence of serum and an increase in cilia length in serum-starved cells [12]. ARP3 is a subunit of the ARP2/3 complex responsible for the nucleation of branched F-actin [9]. Consistently, we reported that overexpression of mir-129-3p, a microRNA, stimulates ciliation in cycling mammalian cells and also increases cilia length by concomitantly downregulating four positive regulators of branched F-actin, ABLIM1, ABLIM3, TOCA1 and ARP2 [13]. ARP2 and TOCA1 are subunit and activator of the ARP2/3 complex [9,15], respectively. ABLIM1 and ABLIM3 are F-actin-associated proteins [16,17] important for lamellipodia formation [13], though
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how the ABLIM proteins affect branched F-actin in lamellipodia is currently unclear. Furthermore, although our results suggest that mir-129-3p needs to target all four proteins to maximize its influence on ciliation, the ablation of any one of them is actually sufficient to phenocopy the effects of the mir-129-3p overexpression. The mir-129-3p overexpression and the cytochalasin D treatment do not display additive effects on either ciliogenic efficiency or cilia length, suggesting that they both affect similar cellular machineries [13]. Therefore, inhibition of branched F-actin formation is a common theme in these studies. These results also provide a reason to serum starvation-induced ciliogenesis because serum starvation is well-known to repress lamellipodia formation [18,19]. The inhibitory effect of the knockdown of Missing-in-Matastasis (MIM) on starvation-induced ciliogenesis [14] may also be attributed to branched F-actin. The ablation of MIM, a monomeric globular actin (G-actin)-binding protein [20], stimulates actin polymerization by enhancing the Src-dependent phosphorylation of Cortactin [14,21]. Cortactin is a class-II nucleation-promoting factor (NPF) of the ARP2/3 complex and also able to promote the formation of branched F-actin together with class-I NPFs such as the WASP family proteins WASP, N-WASP, and SCAR/WAVE [9]. Interestingly, although MIM and Cortactin have been shown to colocalize extensively at the cell cortex [22], a portion of MIM is located at the basal body and this localization appears to be important for its role in ciliogenesis, suggesting the importance of the regional inhibition of actin polymerization in cilia maintenance [14]. Although RNAi, overexpression and serum starvation are not physiological, the changes in actin dynamics are indeed of
Golgi Recycling endosome Plasma membrane protein Ciliary membrane protein Branched F-actin Vesicles Rab11 Rab8 BB (iv)
(ii)
(iii) Inhibition of branched F-actin
(i)
Fig. 1 – A model for roles of branched F-actin in ciliogenesis. (i) Rab8 vesicles are usually sorted from recycling endosomes with the help of branched F-actin and transported by microtubule-dependent motor kinesin (not depicted) to polarized membrane domains such as the lamellipodium. (ii) On the other hand, the accumulation of ciliogenic vesicles including those positive for Rab8, Rabll, and ciliary membrane proteins at the centrosome is critical for efficient ciliogenesis and cilia elongation. (iii) Under ciliogenic conditions, inhibition of branched F-actin impairs the regular vesicle sorting processes, resulting in generation and/or accumulation of ciliogenic vesicles at the centrosome region to promote cilia formation. (iv) The cortical actin network contributes to the cilia membrane domain (CMD) formation by tethering plasma membrane proteins and hence excluding them from the cilium.
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importance in ciliation in vivo. The antagonistic relationship between MIM and Cortactin appears to regulate cilia resorption before M entry because the levels of Cortactin phosphorylation and MIM alter inversely in G2/M phases of the cell cycle [14]. On the other hand, our studies suggest that mir-129-3p is a bona fida positive regulator of ciliation in vertebrate tissues. mir-129-3p is highly expressed in mouse tissues abundant in primary cilia, including brain, retina, kidney and testis. Its inhibition in zebrafish embryos suppresses ciliation in Kupffer’s vesicle and the pronephros, where mir-129-3p is highly expressed, and causes phenotypes typical of ciliapathies, including a curved body, pericardial edema, and defective left-right asymmetry [13]. mir-129-3p also contributes to the serum starvation-induced ciliogenesis because it is moderately upregulated in RPE1 cells after serum starvation and its neutralization inhibited ciliation [13]. Branched F-actin appears to impact ciliogenesis by modulating membrane trafficking. Electron microscopy revealed that a membrane vesicle associated with the distal end of the mother centriole is involved in the initial steps of ciliogenesis and forms subsequently the ciliary sheath [23–25]. Furthermore, cilium constitutes a special membrane domain into which membrane components and membrane proteins need to be delivered from the cytoplasm [3,6,25]. RAB8 is a small GTPase responsible for the vesicle trafficking to the polarized membrane domains such as lamellipodia of migrating cells and the apical side of epithelial cells, whereas RAB11 is important for membrane trafficking from the trans-Golgi network (TGN) and of the recycling endosome (Fig. 1) [26–28]. The ‘‘regular’’ functions of these RABs, however, can be switched to cilia-specific roles because RAB11 recruits and targets Rabin8, the guanine nucleotide exchange factor (GEF) for RAB8, to the centrosome to activate RAB8 for the IFT during ciliogenesis [29,30]. RAB8-positive vesicles becomes abundant at the centrosome area about 1 h after serum withdrawal [29]. Inhibition of branched F-actin by the ablation of ARP3, cytochalasin treatment, or overexpression of mir-129-3p has also been shown to promote the centrosomal accumulations of ciliogenic vesicles positive for Rab11 [12,13]. Therefore, inhibition of branched F-actin is important for the switch from ‘‘regular’’ to ciliogenic trafficking (Fig. 1). Detailed mechanism on how branched F-actin inhibits ciliogenic vesicle trafficking is still not known. Nevertheless, branched F-actin has recently been shown to widely participate in membrane sorting and trafficking by providing constricting forces in the form of a scaffold to facilitate membrane scission [10,31,32]. It is found to exist on different membrane organelles under the control of the WASP family NPFs such as WASH and WHAMM. Inhibition of its nucleation induces marked morphological changes, e.g. tubulation, in various membrane organelles and represses regular membrane trafficking [10,31–34]. RAB8 is enriched in vesicles at the leading edge. Cytochalasin D treatment enhances the formation of Rab8-positive membrane tubules, presumably by disrupting the membrane recycling route of endosomes [27,35]. Therefore, inhibition of branched F-actin possibly results in formation, re-distribution and trafficking of ciliogenic vesicles, including those positive for RAB8 and RAB11 (Fig. 1). In agreement with this postulation, PLA2G3, a phospholipase important for the generation and maintenance of membrane tubules [36–38], is found to serve as a positive regulator of ciliogenesis [12].
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Interestingly, it seems that not all branched F-actin is harmful to cilia because such a form of actin is also important for the selectivity of ciliary components. Periciliary diffusion barrier(s) are found to gate the entry into and exit from the cilia [39]. For instance, a size-exclusion permeability barrier has recently been found to regulate the ciliary concentrations of soluble proteins and other macromolecules using a mechanism analogous to that of the nuclear pore [40]. Furthermore, a disc-like ciliary membrane domain (CMD) excluding cortical F-actin and plasma membrane proteins such as podocalyxin and CEACAM1 also exists at the ciliary base in polarized MDCK cells [41,42]. Further studies indicate that the cortical actin network outside the CMD selectively tethers the membrane proteins depending on specific ‘‘retention signals’’ to exclude them from the CMD [41]. How the CMD lacks F-actin, however, is not clear. Could the basal body-localized MIM [14] hinder cortical actin polymerization there? Kim et al. [12] found in their RNAi screen that the ablation of two Gelsolin family F-actin severing proteins GSN and AVIL [43] leads to ciliogenesis defect, implying that during starvation-induced ciliogenesis certain F-actin-based structure(s) need to be chopped up. If the tethering mechanism is also effective in non-polarized cells, could the servering activity of GSN and AVIL be used to trim the cortical actin from the CMD? There are other unanswered questions on the relationship between cilia and the actin cytoskeleton as well. For instance, the fact that the inhibition of branched F-actin stimulates ciliogenesis also indicates a promotion of the mother centriole-to-basal body transition. The removal of CP110 from the distal end of the mother centriole is believed as an important step for the transition [5]. Although both serum starvation and the overexpression of mir129-3p downregulate CP110 [5,13,44], the cytochalasin D treatment fails to affect the global CP110 levels, nor do Cep97 and Kif24 [13] (data not shown), important for the centrosomal recruitment of CP110 [44,45]. Therefore, how the cytochalasin D treatment leads to the removal of CP110 from the mother centriole [13]remains elusive. Furthermore, inhibition of branched F-actin is not the only factor that impacts ciliogenesis. Serum starvation can generally induce ciliation in more than 80% of RPE1 cells, whereas cytochalasin D treatment or even the overexpression of miR-129-3p to 10000-fold over the endogenous level only stimulated ciliation in 50% of cells [12,13]. It is thus intriguing to elucidate how additional factor(s) synergize with actin dynamics to achieve maximal ciliogenic efficiency. Besides Rabin8, RPGR (retinitis pigmentosa GTPase regulator), a ciliopathy protein required for the ciliary localization of Rab8, also functions as a GEF for Rab8 during ciliogenesis [46]. Interestingly, knockdown of RAB8 or RPGR affects actin dynamics [27,47]. Therefore, it will be interesting to examine whether the RAB8-related changes in actin dynamics also has a role in cilia formation.
Acknowledgements
The authors are grateful to J. Cao for his help in preparing the illustration. This work was supported by the National Basic Research Program of China (2012CB945003 and 2010CB912102) and National Science Foundation of China (30971430).
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