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Opposing Effects of Ndel1 and Α1 Or Α2 on Cytoplasmic Dynein Through

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Opposing Effects of Ndel1 and Α1 Or Α2 on Cytoplasmic Dynein Through 2820 Research Article Opposing effects of Ndel1 and α1orα2 on cytoplasmic dynein through competitive binding to Lis1 Chong Ding1, Xujun Liang2, Li Ma1, Xiaobing Yuan2 and Xueliang Zhu1,* 1Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology and 2State Key Laboratory of Neurobiology, Institute of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China *Author for correspondence ([email protected]) Accepted 26 May 2009 Journal of Cell Science 122, 2820-2827 Published by The Company of Biologists 2009 doi:10.1242/jcs.048777 Summary Lis1 is an essential protein whose insufficiency causes aberrant dynein for Lis1 binding in a dose-dependent manner. neuronal positioning during neocortical development. It is Overexpression of α2 in developing rat brain repressed the believed to regulate both cytoplasmic dynein, a microtubule radial migration of neurons and mitotic progression of minus-end-directed motor, through direct interaction, and neuroprogenitors. By contrast, a Lis1-binding-defective point platelet-activating factor acetylhydrolase (PAF-AH) Ib by mutant, α2E39D, was ineffective in the above assays. These results complexing with the catalytic subunits α1 and α2. Although α1 indicate an antagonistic effect of α1, α2 and Ndel1 for Lis1 and α2 are highly expressed in brain, their deficiencies fail to binding, probably to modulate dynein functions in vivo. They cause brain abnormality. Here, we show that overexpression of also help to explain why brain development is particularly α2 or α1 results in inactivation of dynein characterized by Golgi sensitive to a decrease in Lis1 levels. and endosome dispersion and mitotic delay. Further overexpression of Lis1 or Ndel1, a Lis1- and dynein-binding protein that is also crucial for dynein function, restored Golgi Key words: Nudel, Cytoplasmic dynein, Mitosis, Neuronal migration, and endosome distribution. Biochemical assays showed that α1 Platelet-activating factor acetylhydrolase (PAF-AH) Ib complex, and especially α2, were able to compete against Ndel1 and Vesicle transport Introduction functions as well through direct interactions with both DHC and Journal of Cell Science Haploinsufficiency of Lis1 in humans causes type I lissencephaly, Lis1 (Liang et al., 2004; Liang et al., 2007; Ma et al., 2009; Sasaki a severe congenital disease characteristic of smooth brain surface et al., 2005; Shen et al., 2008; Shu et al., 2004; Stehman et al., owing to deficient neuronal migration during the development of 2007; Yan et al., 2003b). the central nervous system (CNS) (Gupta et al., 2002; Reiner et al., Lis1 also serves as the non-catalytic subunit of platelet-activating 1993). Lis1-deficient mice are embryonic lethal soon after factor acetylhydrolase (PAF-AH) Ib (Hattori et al., 1994b). PAF- implantation. Heterozygous mice exhibit delayed neuronal AHs are enzymes that inactivate PAF by removing its acetyl group. migration and abnormal cortical organization, whereas compound At least three types of PAF-AH: PAF-AH Ib, PAF-AH II and PAF- mice with further reduction of Lis1 manifest severe defects in AH plasma, have been characterized in mammals (Karasawa et al., cortical development (Gambello et al., 2003; Hirotsune et al., 1998). 2003). PAF-AH plasma and PAF-AH II are both monomeric Therefore, the neuronal function of Lis1 is sensitive to its dosage. polypeptides with ~41% sequence identity. By contrast, PAF-AH Why this occurs, however, is not clear. Ib is a heterotrimeric protein complex enriched in brain and testis Lis1, Nde1 (also called NudE), Ndel1 (also called Nudel for (Karasawa et al., 2003; Tjoelker and Stafforini, 2000). α1 (also NudE-like) and cytoplasmic dynein form an evolutionarily named Pafah1b3) and α2 (Pafah1b2), two homologous proteins conserved genetic pathway in eukaryotes (Wynshaw-Boris and sharing ~63% sequence identity, are catalytic subunits of PAF-AH Gambello, 2001). Cytoplasmic dynein is a microtubule (MT)-based Ib, present either in the form of heterodimers or homodimers, and minus-end-directed motor composed of two heavy chains whereas Lis1 (Pafah1b1) is the regulatory subunit (Hattori et al., (DHC) and several intermediate chains (DIC), light intermediate 1994b; Karasawa et al., 2003; Tjoelker and Stafforini, 2000). α2 chains and light chains. It is widely involved in mitosis, intracellular is ubiquitously expressed, with highest expression levels in brain, trafficking, and cell migration (Dujardin et al., 2003; Gomes et al., whereas α1 is predominantly expressed in embryonic brain 2005; Hirokawa, 1998; Hook and Vallee, 2006; Karki and Holzbaur, (Koizumi et al., 2003; Manya et al., 1998). Unlike Lis1, however, 1999). Ndel1, Nde1 and Lis1 probably serve as positive regulators neither α1- nor α2-deficient mice show apparent neurological of dynein. Lis1 binds to dynein and is involved in dynein functions defects. Instead, α2-null or double-knockout mice are sterile in mitosis (Faulkner et al., 2000; Hebbar et al., 2008; Tai et al., because of defective spermatogenesis, whereas α1-null mice have 2002) and cell migration (Dujardin et al., 2003; Tsai et al., 2007). normal fertility possibly because of functional compensation by α2 Lis1 also interacts directly with the N-terminus of Nde1 or Ndel1 (Koizumi et al., 2003; Yan et al., 2003a). (Feng et al., 2000; Niethammer et al., 2000; Sasaki et al., 2000). Since both dynein and the PAF-AH Ib complex share Lis1 as Ndel1 (presumably Nde1 as well) is crucial for a variety of dynein their regulatory factor or subunit, they might exhibit functional Modulation of dynein function 2821 interplay by competing for Lis1, especially when wild-type Lis1 Disruption of dynein activity therefore results in their dispersion levels are attenuated by its gene mutations. In this study, we or fragmentation (Burkhardt et al., 1997; Harada et al., 1998; Liang addressed such questions mainly by overexpressing α1 or α2 to et al., 2004). Indeed, whereas most WGA-positive vesicles were alter the stoichiometry of α-subunits and Lis1. We showed that concentrated at the MTOC in untransfected or GFP-expressing cells excess α1 or α2 can disrupt dynein activity through competitive (Fig. 1A, panels 1-2), they were dispersed in cells overexpressing interaction with Lis1 against dynein and Ndel1 in cultured GFP-α2 (Fig. 1A, panels 3-4). Similar effects were observed for mammalian cells. Moreover, overexpression of α2 in rat brain lysosomes labeled with Lysotracker (data not shown). By contrast, impaired neuronal migration during CNS development. overexpression of a Lis1-binding-defective point mutant (E39D) of Considering the presence of high α1 and α2 levels in brain, such α2 (Yamaguchi et al., 2007) failed to cause vesicle dispersion (Fig. an antagonistic interplay between dynein and PAF-AH Ib might 1A, panels 5-6), indicating a requirement for the Lis1-binding help to explain why Lis1 haploinsufficiency tends to disrupt activity of α2. Similarly, overexpression of GFP-α1 in Cos7 cells neuronal migration. also led to vesicle dispersion, although in a less-potent way (Fig. 1A, panels 7-8). These results indeed suggest inhibition of dynein- Results mediated vesicle transport by excess α1 and α2 in a Lis1-binding- Overexpression of α2 or α1 impairs retrograde vesicle dependent manner. transport α2 overexpression also led to disorganization of MT arrays in To investigate whether the catalytic subunits of PAF-AH Ib were Cos7 cells. Whereas clear radial MT arrays were visible in able to repress dynein functions by sequestering Lis1, we 67.8±2.5% of GFP-positive cells, the value was only 8.7±2.0% overexpressed α2 in Cos7 cells and examined the distribution of in cells overexpressing GFP-α2 (Fig. 1C-D). Nevertheless, the membrane organelles labeled with TRITC-conjugated wheatgerm vesicle dispersions were not fully correlated with MT agglutinin (WGA). WGA recognizes glycoproteins at the plasma organizations because WGA-positive vesicles were also found to membrane, endosome, and trans-Golgi cisternae (Liang et al., 2004; be dispersed in GFP-α2-expressing cells with radial MT arrays Raub et al., 1990; Virtanen et al., 1980). As endosomes and Golgi (Fig. 1E), possibly owing to different sensitivities of the MT cisternae are subjected to dynein-mediated transport towards MT organization and vesicle trafficking to dynein inactivation. As the minus-ends, they are usually enriched around the MT-organizing dynein motor is important for MT organization (Echeverri et al., center (MTOC) (Hirokawa, 1998; Karki and Holzbaur, 1999). 1996; Malikov et al., 2004; Quintyne et al., 1999), the influence Journal of Cell Science Fig. 1. Effects of α2 or α1 overexpression on vesicle distribution and MT organization. (A,B) Incidence of vesicle dispersion in Cos7 cells transiently overexpressing GFP or the indicated GFP-tagged proteins. Cells were fixed in methanol and labeled with TRITC-WGA. Vesicles labeled with WGA are indicated by arrows in transfectants or arrowheads in untransfected cells. The insets in panels 3 and 7 show nuclear staining of corresponding transfectants. Error bars show s.d. **P<0.01 in Student’s t-tests. (C,D) MT organization in Cos7 cells overexpressing GFP or GFP-α2. Arrows indicate MT arrays in transfectants. Inset shows nuclear staining. Error bars indicate s.d. **P<0.01 in Student’s t-test. (E) Vesicle dispersion is seen in GFP-α2- positive cells with either abnormal (large arrows) or normal (small arrows) MT organization. Arrowheads indicate MT and vesicle organization in untransfected cells. 2822 Journal of Cell Science 122 (16) of α2 overexpression on MT organization is also consistent with α2 and α1 can compete against dynein for Lis1 inactivation of dynein. Nevertheless, unlike CHO cells To further corroborate the above results, we examined whether α2 (Yamaguchi et al., 2007), Cos7 and HEK293T cells overexpressing or α1 overexpression was indeed able to sequester Lis1 from dynein. either α2 or α1 did not form pleiomorphic nuclei (Fig. 1A,C,E, FLAG-Lis1 was coexpressed in HEK293T cells with GFP, GFP-α1, insets; also see Fig.
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