The Mammalian Golgi Regulates Numb Signaling in Asymmetric Cell Division by Releasing ACBD3 During Mitosis

The Mammalian Golgi Regulates Numb Signaling in Asymmetric Cell Division by Releasing ACBD3 during Mitosis Yan Zhou,1 Joshua B. Atkins,1,2 Santiago B. Rompani,1,3 Daria L. Bancescu,1 Petur H. Petersen,1,4 Haiyan Tang,1,2,5 Kaiyong Zou,1 Sinead B. Stewart,1 and Weimin Zhong1,2,* 1 Department of Molecular, Cellular, and Developmental Biology 2 Interdepartmental Neuroscience Program Yale University, P.O. Box 208103, New Haven, CT 06520, USA 3 Present address: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. 4 Present address: Center for Molecular Biology and Neuroscience, University of Oslo, Oslo, Norway. 5 Present address: George Washington University Law School, Washington, DC 20052, USA. *Correspondence: [email protected] DOI 10.1016/j.cell.2007.02.037 SUMMARY Drosophila melanogaster, for example, sensory organ precursor (SOP) cells divide asymmetrically to produce Mammalian neural progenitor cells divide a IIA and a IIB cell (Gho et al., 1999). Drosophila Numb asymmetrically to self-renew and produce (d-Numb), a cytosolic signaling protein, distributes sym- a neuron by segregating cytosolic Numb pro- metrically during interphase but becomes localized to teins primarily to one daughter cell. Numb sig- only one-half of the cell membrane after the SOP cell en- naling specifies progenitor over neuronal fates ters mitosis and, consequently, is segregated primarily but, paradoxically, also promotes neuronal into the IIB cell (Rhyu et al., 1994). This asymmetric d-Numb distribution is essential for distinguishing the differentiation. Here we report that ACBD3 is two daughter cells; d-Numb loss causes both daughter a Numb partner in cell-fate specification. cells to become IIA, whereas its symmetric inheritance ACBD3 and Numb proteins interact through an yields two IIB cells. Interestingly, d-Numb loses its ability essential Numb domain, and the respective to specify cell fates shortly after mitosis; although the loss- and gain-of-function mutant mice share IIA fate requires its initial absence, newly synthesized phenotypic similarities. Interestingly, ACBD3 d-Numb later accumulates in IIA cells, which then segre- associates with the Golgi apparatus in neurons gate the protein asymmetrically to produce a hair and and interphase progenitor cells but becomes a socket cell. d-Numb is similarly used by a variety of cytosolic after Golgi fragmentation during mito- neural and nonneural precursor cells to generate cellular sis, when Numb activity is needed to distinguish diversity during development (reviewed in Betschinger the two daughter cells. Accordingly, cytosolic and Knoblich, 2004; Prokopendo and Chia, 2005). Mammalian Numb homologs are encoded by two ACBD3 can act synergistically with Numb to genes, Numb (m-numb) and numblike (Numbl or nbl; Sal- specify cell fates, and its continuing presence cini et al., 1997; Zhong et al., 1996, 1997). The importance during the progenitor cell cycle inhibits neuron of Numb-mediated asymmetric cell division in mammalian production. We propose that Golgi fragmenta- development is most clearly demonstrated in the embry- tion and reconstitution during cell cycle dif- onic nervous system, where such divisions are used by ferentially regulate Numb signaling through progenitor cells to balance the competing needs of self- changes in ACBD3 subcellular distribution and renewal and neuron production during the extended represent a mechanism for coupling cell-fate period of neurogenesis. Direct imaging experiments specification and cell-cycle progression. show that mammalian neural progenitor cells behave like stem cells and can divide asymmetrically to generate a daughter progenitor cell (self-renewal) and a neuron INTRODUCTION (Chenn and McConnell, 1995; Noctor et al., 2004; Qian et al., 1998). The m-Numb protein localizes asymmetri- Cell-fate determination is often precisely coordinated with cally in neural progenitor cells and can be segregated to cell-cycle progression. This is most apparent during an only one daughter cell (Cayouette and Raff, 2003; Shen intrinsically asymmetric cell division, which produces et al., 2002; Zhong et al., 1996, 1997). Loss-of-function two daughter cells that are already different at birth. In studies using mice show that m-numb and Numbl are Cell 129, 163–178, April 6, 2007 ª2007 Elsevier Inc. 163 redundant during neurogenesis, but inactivation of both tion. We also show that the process of Golgi fragmentation genes causes progenitor cells to deplete prematurely and reconstitution regulates the subcellular distribution throughout the embryonic nervous system due to an initial of ACBD3 and likely represents a novel mechanism for overproduction of neurons at the expense of progenitor differentially activating intracellular signaling at different cells (Petersen et al., 2002, 2004). These findings point phases of the cell cycle. to a role for mammalian Numb proteins in enabling neural progenitor cells to balance self-renewal and neuron pro- RESULTS duction by segregating asymmetrically to the progenitor daughter cells to specify their fates. However, m-Numb ACBD3 Is a Novel Numb-Binding Protein and Numbl proteins quickly appear in the neuronal daugh- To elucidate the Numb-signaling pathway, we used yeast ter cells (Zhong et al., 1997), and their presence is essen- two-hybrid screen to identify mouse proteins that physi- tial for neuronal differentiation (Huang et al., 2005; cally interact with m-Numb and identified a clone (MERRY Petersen et al., 2002). Thus, Numb signaling seems to MAGPIE) encoding an evolutionarily conserved protein play two contradictory roles: namely, it promotes progen- (Figure 1A). This protein contains a putative acyl- itor over neuronal fates but is also required for neuronal coenzyme A-binding domain and was later named ACBD3 differentiation. In other words, the ability of mammalian by the genome project. It was also identified as GCP60 Numb homologs to specify cell fates also appears to be (Sohda et al., 2001) and PAP7 (Li et al., 2001) due to its limited to during and/or shortly after mitosis. ability to bind Giantin, a Golgi membrane protein, and a A major contributor to this coordinated process of cell- peripheral-type benzodiazepine receptor, respectively. fate determination and cell-cycle progression is the cellu- Deletion analysis revealed that the N-terminal 23 amino lar machinery that asymmetrically localizes Numb proteins acids of m-Numb contained the ACDB3-binding (AB) do- only during mitosis (reviewed in Betschinger and Knoblich, main; other m-Numb regions, including the phosphotyro- 2004; Prokopendo and Chia, 2005). Asymmetric localiza- sine-binding (PTB) domain, were incapable of interacting tion, however, is not necessary for Numb to specify cell with ACBD3 (Figures 1B and 1D). The AB domain is highly fates; Numb proteins that distribute uniformly in the cyto- conserved between mouse and d-Numb proteins (Fig- sol are fully functional, although they force the same fates ure 1B), and ACBD3 could also bind to Numbl and upon the two daughter cells (Knoblich et al., 1997; Zhong d-Numb (Figure 1C). As expected, a fusion protein of et al., 1997). Thus, the asymmetric localization machinery Glutathione-S-Transferase (GST) and the C-terminal 366 can explain how the two daughter cells become different amino acids of ACBD3 (ACBD3-C), but not the ACB do- but not why Numb proteins lose the ability to specify fates main (ACBD3-N), could copurify m-Numb from mouse after division, raising the possibility that Numb signaling brain extracts (Figure 1E). We also generated ACBD3 itself is also regulated during cell cycle. The components antibodies (Figure 2B) and performed coimmunoprecipi- of Numb signaling, however, remain largely unknown, par- tation experiments to examine whether ACBD3 and ticularly in mammals. Drosophila studies show that Numb m-Numb interact in vivo. Indeed, in extracts from HeLa acts by inhibiting the transmembrane receptor Notch (Guo cells cotransfected with plasmids expressing m-Numb et al., 1996; Spana and Doe, 1996), which mediates cell- and a myristoylated form of ACBD3 (ACBD3Myr; see Fig- cell communication in many contexts during metazoan ure 5A below), an m-Numb antibody, but not an unrelated development (reviewed in Artavanis-Tsakonas, 1999). Be- CSN1 antibody (Yang et al., 2002), was able to coprecipi- cause Numb also specifies many unrelated fates, it has tate both the endogenous ACBD3 and the exogenous been postulated that asymmetric Numb presence simply ACBD3Myr (Figure 1F). enables the daughter cells to choose differently between two fate options by biasing Notch-mediated cell-cell com- The ACDB3-Interacting Domain Is Essential munication (reviewed in Zhong, 2003). Whether and how for Numb Activity this antagonistic Numb-Notch relationship is conserved Interestingly, whereas the N-terminal PTB domain (Fig- in mammals, however, remain unclear (Petersen et al., ure 1B) is essential for Numb activity (Knoblich et al., 2006). 1997; Yaich et al., 1998), the first 23 amino acids compris- In mammalian cells, cell-cycle progression is accompa- ing the AB domain are not part of the PTB (Li et al., 1998). nied by dramatic changes in the morphology of the Golgi Further deletion analysis revealed that neither the first apparatus (reviewed in Altan-Bonnet et al., 2004; Colanzi 7 amino acids of the AB domain nor the last 16 could et al., 2003; Shorter and Warren, 2002). During interphase, mediate binding to ACBD3 by themselves. Surprisingly, mammalian Golgi membranes are organized into intercon- although

Load more