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Formins are conserved proteins that assemble unbranched actin filaments in a regulated, localized manner. Budding yeast's two formins, Bni1p and Bnr1p, assemble actin cables necessary for polarized cell growth and organelle segregation. Here we define four regions in Bni1p that contribute to its localization to the bud and at the bud neck. The first (residues 1–333) requires dimerization for its localization and encompasses the Rho-binding domain. The second (residues 334–821) covers the Diaphanous inhibitory–dimerization–coiled coil domains, and the third is the Spa2p-binding domain. The fourth region encompasses the formin homology 1–formin homology 2–COOH region of the protein. These four regions can each localize to the bud cortex and bud neck at the right stage of the cell cycle independent of both F-actin and endogenous Bni1p. The first three regions contribute cumulatively to the proper localization of Bni1p, as revealed by the effects of progressive loss of these regions on the actin cytoskeleton and fidelity of spindle orientation. The fourth region contributes to the localization of Bni1p in tiny budded cells. Expression of mislocalized Bni1p constructs has a dominant-negative effect on both growth and nuclear segregation due to mislocalized actin assembly. These results define an unexpected complexity in the mechanism of formin localization and function.
The ability to polarize is a universal feature of eukaryotic cells that allows them to grow and divide, move, transport nutrients across epithelia, and so on. To probe the general mechanisms underlying cell polarity, we exploit the budding yeast Saccharomyces cerevisiae as an experimentally accessible model to study polarized growth and organelle segregation during the cell cycle. During bud growth, polarized actin cables are assembled that guide the transport of secretory vesicles for cell growth, as well as the segregation of organelles, all of which depend on transport by the myosin-V motor Myo2p (Pruyne et al., 2004b ). The polarized actin cables are nucleated and elongated by localized formins.
Formins are a family of conserved proteins that assemble unbranched actin filaments. They have been implicated in the formation of multiple cellular structures such as stress fibers, filopodia, cell–cell junctions, phagocytic cups, and contractile rings (Chesarone et al., 2010 ). In budding yeast, there are two formin proteins, Bni1p and Bnr1p, which have different localizations and contribute to different sets of actin cables (Pruyne et al., 2004a ). During bud growth, Bni1p localizes dynamically to the bud tip, where it nucleates the assembly of cables, and later it is found at the bud neck, where it contributes to assembly of the contractile ring prior to cytokinesis, whereas Bnr1p is stably associated with the bud neck to generate cables extending into the mother cell (Pruyne et al., 2004a ; Buttery et al., 2007 ). Although single deletion of either BNI1 or BNR1 is viable, deleting both is lethal (Vallen et al., 2000 ; Ozaki-Kuroda et al., 2001 ). All formins possess the defining domain formin homology 2 (FH2), generally in the C-terminal half of the molecule. Studies with the FH2 domain of Bni1p were the first to show that formins can nucleate the assembly of unbranched actin filaments and then remain associated with the elongating barbed end of the growing filaments (Pruyne et al., 2002 ; Sagot et al., 2002 ). N-Terminal to the FH2 domain is the proline-rich formin homology 1 (FH1) domain, which binds to profilin–actin to enhance the assembly rate at the FH2-associated actin filament end (Sagot et al., 2002 ). As members in the Diaphanous-related formin subfamily, Bni1p and Bnr1p also have an N-terminal Diaphanous inhibitory domain (DID) domain, which has been suggested to bind the C-terminal Diaphanous autoregulatory domain (Li and Higgs, 2003 , 2005 ; Wang et al., 2009 ). This autoinhibitory interaction is relieved at least in part by binding of Rho GTPases to the GTPase-binding domain (GBD), which partially overlaps with the DID (Li and Higgs, 2003 , 2005 ; Lammers et al., 2005 ; Otomo et al., 2005 ; Rose et al., 2005 ; Nezami et al., 2006 ; Seth et al., 2006 ). Biochemical and structural analyses have shown that the N-terminal region of the formin mDia1 is dimerized through a dimerization domain (DD; Li and Higgs, 2005 ; Otomo et al., 2005 ), which is also present in Bni1p. Bni1p contains a unique region, the Spa2p-binding domain (SBD), which binds to Spa2p and has been suggested to localize Bni1p (Fujiwara et al., 1998 ). Spa2p is part of a 12S complex called the polarisome that also contains Pea2p and Bud6p (Sheu et al., 1998 ; Shih et al., 2005 ). Bud6p binds the C-terminal region of Bni1p to enhance actin assembly and contribute to spindle orientation (Fujiwara et al., 1998 ; Moseley and Goode, 2005 ; Delgehyr et al., 2008 ).
The nucleus is a critical organelle whose segregation during cell division is essential for growth. During cell division the nuclear membrane does not break down, so the nucleus must be accurately oriented and elongated relative to the bud neck in order for both the mother and the bud to each inherit one nucleus at the end of mitosis. This nuclear orientation and elongation depends on forces acting on cytoplasmic microtubules radiating from the spindle pole bodies embedded in the nuclear membrane. Early during bud formation, the nucleus is initially oriented by the class V myosin Myo2p, which links Kar9p to Bim1p on the plus end of cytoplasmic microtubules and transports them down actin cables toward the bud (Beach et al., 2000 ; Yin et al., 2000 ; Hwang et al., 2003 ). Because this step requires actin cables nucleated by Bni1p, bni1 mutants have nuclear movement and microtubule orientation defects (Fujiwara et al., 1999 ; Lee et al., 1999 ; Theesfeld et al., 1999 ). Later during anaphase, the elongation of the nucleus depends on the microtubule motor protein Dyn1p and associated dynactin complex (Li et al., 1993 ; Muhua et al., 1994 ). Thus nuclear movement is mediated by two pathways: one through Kar9p/Myo2p/Bni1p, and the other through Dyn1p and components of the dynactin complex. Failure of either pathway is not lethal and results in minor abnormalities in nuclear segregation; however, loss of both can be lethal (Fujiwara et al., 1999 ; Miller et al., 1999 ; Tong et al., 2001 ).
In this study, we dissect Bni1p to define four independent localization regions in the protein. These regions contribute cumulatively to the appropriate localization of Bni1p to influence polarized growth and spindle orientation. We also show that Bni1p constructs that do not localize appropriately have a dominant phenotype that slows growth and interferes with spindle orientation due to the inappropriate assembly of mislocalized actin.
It was shown that the N-terminal 1–1240 amino acids of Bni1p are sufficient for its localization (Ozaki-Kuroda et al., 2001 ). To see whether a smaller piece of Bni1p can still localize appropriately, we expressed, using a GAL1 promoter on a CEN plasmid, a series of C-terminally green fluorescent protein (GFP)–tagged constructs encompassing decreasing regions of the N-terminal domain to narrow down localization determinants. We designed the pieces to be expressed based on homology to structurally defined domains of mDia1 (Otomo et al., 2005 ; Nezami et al., 2006 ; Figure 1A). The constructs were expressed in both bni1Δ (Figure 1, B–O) and BNI1 (Supplemental Figure S1A) cells to test whether the localization regions depend on endogenous Bni1p. All constructs expressed proteins of the expected size (Supplemental Figure S1B). The localizations of different constructs described in the following sections are summarized in Table 1.
Full-length Bni1p-GFP localizes at the bud growth site (Figure 1B), concentrates at the bud tip of small-budded cells, distributes more broadly as a cap in medium-budded cells, disappears from the bud cortex of large-budded cells, and reappears at the bud neck prior to cytokinesis (Ozaki-Kuroda et al., 2001 ; Pruyne et al., 2004a ). Live-cell imaging of the Bni1p–1-1239–GFP construct showed a broader crescent localization than full-length Bni1p at the bud cortex of small and medium-sized buds and also localized to the bud neck late in the cell cycle (Figure 1C). The shortest N-terminal region that localized to the bud cortex well was Bni1p–1-717–GFP (Figure 1D). Because the SBD lies outside this construct and the Bni1p–822-992–GFP construct covering the SBD localizes mostly to the nucleus but also weakly to the bud cortex and bud neck (Figure 1E), Bni1p has at least two localization domains in its N-terminal 1–1239 amino acid region. Constructs shorter than Bni1p–1-717–GFP localized very weakly, with barely detectable localization to the bud cortex and neck (Figure 1, F and H).
As indicated, Bni1p–1-717–GFP localizes to the bud cortex and the bud neck, yet Bni1p–1-596–GFP does so very weakly (Figure 1F). The difference between these two constructs is the region homologous to the DD. Because the DD region of mDia1 has been shown to facilitate dimerization (Li and Higgs, 2005 ), we asked whether dimerization might be important for localization. We tested whether artificially dimerizing the Bni1p–1-596 construct would allow it to localize appropriately. To do this, we appended a leucine zipper from human CREB, which functions as a dimerization domain (Schumacher et al., 2000 ). The leucine zipper fused to GFP is cytoplasmic (Figure 1J), whereas the Bni1p–1-596–leucine-zipper construct localized robustly and broadly to the cell cortex and neck (Figure 1G). With this result in hand, we explored how much of the N-terminal domain, when dimerized, is necessary for this localization. We found that Bni1p–1-333–GFP does not localize to the bud cortex, whereas Bni1–1-333– leucine-zipper localizes to both the bud cortex and the bud neck (Figure 1, H and I). In addition, when the Bni1p–1-596 construct, which does not localize, is fused to a region (717–834) downstream of the DD domain, localization is restored (construct ΔDD 1-834, Figure 1K). Therefore the 717–834 region, which is predicted to form a coiled coil, might also contribute to the dimerization of the N-terminus of Bni1p.
Our results define two regions of Bni1p that localize independently, namely residues 1–333 and 822–992. We next sought to determine whether the region between these two also contributes to localization. To our surprise, Bni1p–334-821–GFP, which covers the DID, DD, and the following predicted coiled-coil (CC) region before the SBD domain, was also able to localize to the bud cortex and bud neck (Figure 1L), similar to Bni1p–1-1239–GFP.
All the localizations were unchanged in BNI1 cells (Supplemental Figure S1A), indicating that localization does not require dimerization with endogenous Bni1p. Thus three distinct localization determinants must exist in the bud and later at the neck. Moreover, the localization of Bni1p–1-333–leucine-zipper, Bni1p–334-821, or Bni1-GFP to the bud cortex is independent of the actin cytoskeleton, as they were unchanged when the actin cytoskeleton was completely disassembled by treating cells for 10 min with 200 μM of the actin-depolymerizing drug latrunculin A (Figure 1, M–O).
Thus Bni1p is localized to the bud cortex by three localization regions, which we will refer to as LD1, LD2, and LD3. LD1 resides in the N-terminal 333 amino acids containing the GBD and requires dimerization. LD2 resides in the 334–821 amino acids covering the DID-DD-CC domains. LD3 resides in the region of the Spa2p-binding domain. As we will discuss, Bni1p has a fourth localization region, LD4, encompassing residues 1231–1953.
To explore how important the localization domains are for growth, we made a series of constructs deleting progressively larger internal regions from Bni1p residue 79 up to the FH1 domain (Figure 2A). These constructs were targeted to the chromosomal locus so that the proteins were expressed as the sole copy from the endogenous BNI1 promoter. Cells with these constructs expressed the appropriately sized proteins and were all viable (Figure 2, B and D). Moreover, growth at 25°C was unaffected by simultaneous deletion of BNR1 (Figure 2B), in support of our earlier finding that yeast are viable expressing just the FH1–FH2 fragment of Bni1p as their sole formin protein (Gao and Bretscher, 2009 ). For ease of description, we have grouped the internal deletion series into two classes: class I deletions do not noticeably affect cell growth, whereas class II deletions, represented by the two largest internal deletions, bni1Δ79-988-3GFP and bni1Δ79-1230-3GFP, grew less well in both BNR1 and bnr1Δ backgrounds (Figure 2B). In fact, class II internal deletions in the BNR1 background grew less well than BNR1 bni1Δ cells.
We next examined the actin cytoskeleton and the localization of the Bni1p-derived constructs C-terminally tagged with 3GFP (Figure 2C and Table 2). In wild-type cells during bud growth, actin cables extend along the surface of the bud and into the mother cell, whereas actin patches concentrate in the bud. Phalloidin staining of class I BNI1 deletion mutants reveals a modest disruption of their actin cytoskeleton, which becomes more noticeable with increasing size of the internal deletion. In the class I construct with the largest deletion, bni1-Δ79-821-3GFP, the actin patches are somewhat polarized, and the actin cables are more disoriented and shorter, especially in the mother cells. Localization of the class I BNI1 mutant proteins showed that in general they localized very similarly to wild-type Bni1p-3GFP, namely concentrating at the nascent bud site and the tips of small buds, in a crescent over the cortex of medium buds, and at the septal plane between dividing cells (Figure 2C). Despite this general similarity, bni1Δ79-821-3GFP localizes to the nucleus and less robustly to the cell cortex. However, in both class II BNI1 mutants, actin cables are obviously short and disoriented, and actin patches are severely depolarized. This localization is independent of the first 78 amino acids, as bni1Δ1-1230 shows the same localization (Figure 2C). The localization of the class II BNI1 mutants was much weaker, being seen in a polarized manner only in unbudded and tiny budded cells. The localizations of these Bni1p mutants are summarized in Table 2.
Because the constructs expressing just the FH1-FH2-COOH of Bni1p are localized at the beginning of the cell cycle, an additional localization determinant must exist in this region. This localization was also unchanged in cells treated with latrunculin A for 10 min (Supplemental Figure S2), showing that, in addition to the three N-terminal localization regions just documented, Bni1p has a fourth F-actin-independent localization region in the FH1-FH2-COOH part of Bni1p, namely LD4. No bud neck localization was observed for LD4, although note that these cells are depolarized and the morphological defect may affect neck recruitment (Figure 2C).
The defining difference between the class I and class II internal deletions of Bni1p is the presence or absence of the SBD. If Spa2p is an important determinant for Bni1p localization, one would predict that combining spa2Δ with class I BNI1 mutants should phenocopy a class II mutant. Indeed, combining class I mutants with spa2Δ compromised the growth of the cells in a progressive manner, with the larger deletions being more affected (Figure 3A). To explore the basis for these growth defects, the actin cytoskeleton and localization of the Bni1p-3GFP truncated proteins were examined. When spa2Δ was combined with the class I mutants, actin structures were more disrupted, especially in bni1Δ79-821 spa2Δ cells, which had greatly reduced cables and depolarized actin patches. The localization of the Bni1p-deletion constructs was also affected, with the larger deletions being the most severe (Figure 3B).
We next examined the effect of combining spa2Δ with the class II BNI1 deletion mutants, which lack the SBD, and found, unexpectedly, that they show synthetic lethality (Figure 3C). Thus, although the SBD is not present in these Bni1p constructs, Spa2p is essential for the viability of these cells, indicating that Spa2p has an important function independent of its interaction with the SBD of Bni1p.
We recently reported that yeast is viable when the only formin present is a construct of just the FH1 plus FH2 domains of either Bni1p or Bnr1p (Gao and Bretscher, 2009 ). This implies that the N-terminal localization regions described earlier are not essential for cell growth. To study the role of N-terminal localization regions in Bni1p's function, we sought a more stringent condition for cell growth. It has been reported that spindle orientation, necessary for proper nuclear segregation, requires either the Kar9p/Myo2p/Bni1p pathway or the dynein/dynactin pathway, with either being dispensable for spindle orientation, but defects in both being lethal (Fujiwara et al., 1999 ; Miller et al., 1999 ; Tong et al., 2001 ). The Kar9p/Myo2p/Bni1p pathway has been shown to involve the binding of Myo2p to Kar9p on the ends of cytoplasmic microtubules for transport along Bni1p-nucleated actin cables into the bud (Yeh et al., 2000 ; Yin et al., 2000 ; Hwang et al., 2003 ). Because Arp1p is a core component of the dynactin complex, disruption of ARP1 leads to the disassembly of the dynactin complex (Schroer, 2004 ). Thus in arp1Δ cells, actin cables nucleated and oriented by localized Bni1p should be essential for spindle orientation and thereby provide a genetic system to study the role of the localization regions of Bni1p in its function.
When we combined bni1Δ with arp1Δ we were surprised to find that a bni1Δ arp1Δ (and a bni1Δ dyn1Δ; Supplemental Figure S3A) strain is viable, thereby in principle compromising the design of our study and supporting alternative observations of others (Lee et al., 1999 ; Yeh et al., 2000 ). Fortuitously, when we combined arp1Δ with the class I and class II series of internal deletions in Bni1p we uncovered a clear finding: class I deletions are viable in conjunction with arp1Δ, whereas class II deletions are inviable in conjunction with arp1Δ (Figure 4A).
To explore the potential role of the Bni1p localization determinants in spindle orientation, we examined the class I mutants in more detail in both ARP1 and arp1Δ cells. Because these processes require microtubules that are less stable at low temperature, we examined the sensitivity of the strains for growth at 14°C and in the presence of sublethal levels of the microtubule-destabilizing drug benomyl (Figure 4B). In ARP1 cells, class I mutants like bni1Δ showed no cold or benomyl sensitivity (Figure 4B). However, in the arp1Δ background, the largest internal deletion of class I mutants, bni1Δ79-821, shows more cold and benomyl sensitivity than bni1Δ arp1Δ cells (Figure 4B), suggesting that class I truncations are at least somewhat compromised in microtubule-mediated spindle orientation. Consistent with this phenotype, DNA staining of class I truncations in ARP1 cells revealed that they have a modest defect in nuclear segregation at 16°C, similar to a bni1Δ cell, and this defect is strongly exacerbated in arp1Δ cells (Figure 4C). The binuclear phenotype, as well as cold and benomyl sensitivity, suggests that BNI1 class I mutants have defects in spindle orientation. Indeed, the spindle orientation in small- to medium-budded cells was observed using GFP-Tub1p and confirmed that Bni1p class I mutants exacerbate the spindle orientation defects seen in arp1Δ cells (Figure 4D).
Class II mutants in ARP1 cells are cold sensitive and benomyl sensitive by themselves, displaying more severe microtubule-related defects than bni1Δ cells (Figure 4E), indicating that they have a negative gain of function in spindle orientation. Consistent with this interpretation, heterozygous BNI1/class II mutants in arp1Δ/arp1Δ cells also exhibited greater cold and benomyl sensitivity than BNI1/bni1Δ arp1Δ/arp1Δ cells (Figure 4F).
Because the essential role of Bni1p in S. cerevisiae is to nucleate and assemble actin cables, we asked whether the dominant phenotypes caused by the class II Bni1p mutants are related to their ability to assemble actin filaments at inappropriate locations. We first tested this model by mutating the FH2 domain of a class II truncation by changing a single amino acid (I1431A) that has been reported to abolish actin assembly activity (Xu et al., 2004 ). Expression of bni1Δ79-988 with the I1431A mutation is not synthetically lethal with arp1Δ and is not cold sensitive, behaving similarly to a bni1Δ mutation (Figure 5, A and B). Thus the deleterious phenotypes caused by bni1Δ79-988 are due to its actin assembly activity.
Because the class II construct Bni1Δ79-988p–3GFP is poorly localized (Figure 2C), we explored whether the growth defects are due to mislocalized actin assembly. To test this, we fused the Cdc42-Rac interactive binding (CRIB) domain of Gic2p to the N-terminus of this class II mutant. Gic2p is a Cdc42p effector that is localized to the bud tip during bud growth and bud neck during cytokinesis by binding through its CRIB domain to Cdc42-GTP (Burbelo et al., 1995 ; Brown et al., 1997 ). The addition of the CRIB domain to Bni1pΔ79-988 largely restored its localization to the bud cortex and also rescued the cold sensitivity (Figure 5, C and D). In addition, phalloidin staining showed polarized actin patches and directed actin cables in CRIB-bni1Δ79-988 cells (Figure 5D). Therefore the deleterious phenotypes associated with class II mutants are a result of their ability to assemble mislocalized actin filaments.
To perform their physiological functions in actin assembly, formins need to be appropriately localized. Despite this critical aspect, remarkably little is known about how they are localized. It is clear for the mDia proteins that the N-terminal region is important for localization (Seth et al., 2006 ; Brandt et al., 2007 ; Gorelik et al., 2011 ). In the recent study of Gorelik et al. (2011) , a basic domain N-terminal to GBD was found to be important for plasma membrane localization, and the small GTPase Rif could also contribute significantly, implying that these two factors work together for correct localization.
We took this type of analysis one step further in our identification of regions in Bni1p involved in its localization and sought to understand the functions of the identified localization domains. This analysis showed an unexpected complexity by revealing that Bni1p has at least four independent localization domains that can target the protein to the cell cortex in a manner independent of either endogenous Bni1p or filamentous actin. Three of these localization domains reside in the N-terminal 1200 residues of the protein and are independent of an actin cytoskeleton. LD1 (residues 1–333) coincides well with a region homologous to the structurally defined GBD domain. Formins of the Diaphanous family are regulated by binding to active Rho proteins, and this binding occurs through both the GBD region and the DID (Rose et al., 2005 ). The need for dimerization probably reflects a low affinity of the isolated domain for the cell cortex, so that only with the higher affinity of a dimer is the interaction clear. Native Bni1p is presumably a dimer, as the DD domain, as well as the FH2 domain, can dimerize (Moseley et al., 2004 ; Xu et al., 2004 ; Otomo et al., 2005 ). The second localization domain, LD2 (residues 334–834), includes the DID, the DD, and a region predicted to form a coiled coil. By virtue of the DD and predicted coiled-coil domain, this region almost certainly dimerizes. This region seems to be the most robust localization region, as the internal deletion constructs retaining it localize well in both SPA2 and spa2Δ cells, whereas those lacking it localize poorly, especially in spa2Δ cells. Finally, LD3 encompassing the SBD has been identified as being important for Bni1p localization (Fujiwara et al., 1998 ; Ozaki-Kuroda et al., 2001 ). We identified all of these localization domains in cells in which the constructs were overexpressed. The fact that they localized suggests that the factors that localize them to the bud cortex or bud tip are likely to be much more abundant than endogenous Bni1p. In support of this conclusion, attempts to disrupt the function of endogenous Bni1p by expression of N-terminal constructs failed to cause any obvious phenotype. In addition to the three N-terminal localization domains, we found that the complementary C-terminal part LD4 (1231–1963), comprising the FH1, FH2, and C-terminal region, also has a weak localization domain to incipient and small buds. This localization is independent of either endogenous Bni1p or the presence of filamentous actin.
Having identified four localization domains in Bni1p, we sought to investigate their functions. Our original strategy assumed that correct localization is an essential part of formin function, so a deletion analysis of Bni1p regions in a bnr1Δ cell should reveal the essential localization domain. Accordingly, we made a series of N-terminal deletions in the chromosomal copy of Bni1p and assessed their effect on growth in BNR1 and bnr1Δ cells. During this analysis, we made the unexpected discovery that yeast can grow remarkably well in the absence of a localized formin, namely when the only formin protein present was the delocalized FH1 plus FH2 domain of either Bni1p or Bnr1p (Gao and Bretscher, 2009 ). Despite this result, the N-terminal deletions of Bni1p were very informative. Deleting up to the SBD had little effect on growth, whereas deleting more of the N-terminal region to remove the SBD was deleterious to growth and resulted in quite depolarized cells that, even in the presence of Bnr1p, conferred a more severe phenotype than bni1Δ. Expression of this region of Bni1p therefore confers a dominant-negative phenotype.
Before this study, the only identified localization mechanisms for Bni1p were provided by Spa2p through the SBD (Fujiwara et al., 1998 ; Ozaki-Kuroda et al., 2001 ) and potentially through the polarisome-associated Bud6p to the C-terminal region (Ozaki-Kuroda et al., 2001 ). Because N-terminal deletion constructs containing the SBD were relatively healthy, whereas those lacking it were dominant negative, we suspected that combining spa2Δ with our Bni1p constructs containing the SBD should phenocopy those lacking it. spa2Δ indeed compromised growth of Bni1p constructs with the SBD. It is surprising that the dominant-negative Bni1p constructs that lack the SBD are synthetically lethal with spa2Δ, indicating that Spa2p performs a function in addition to binding the SBD of Bni1p. One possibility is that Spa2p is still contributing to Bni1p localization through Bud6p, which binds both Spa2p and the C-terminal region of Bni1p (Evangelista et al., 1997 ; Sheu et al., 1998 ; Moseley and Goode, 2005 ).
Because Bni1p localization is not essential for viability in bnr1Δ cells, we explored the possibility of exploiting a situation in which Bni1p localization is important even in the presence of Bnr1p. Spindle orientation is required for proper nuclear segregation and depends both on the Bni1p/Myo2p/Kar9p pathway and the dynactin/dynein pathway. In the absence of the dynein/dynactin pathway, Bni1p/Myo2p/Kar9p becomes the critical mechanism for spindle orientation. We therefore combined our N-terminal Bni1p deletion series with arp1Δ cells, lacking a component of the dynactin complex. Indeed, we found a modest spindle orientation defect (as determined by benomyl sensitivity and the fraction of mother cells with two or more nuclei) in the mutants even in ARP1 cells that increased as additional localization domains were deleted from the N-terminal region of Bni1p, and these defects were strongly enhanced in arp1Δ cells. Therefore all the identified N-terminal localization domains contribute to the Bni1p-dependent spindle orientation pathway. As described earlier, Bni1p constructs from which all the N-terminal localization domains were deleted had a dominant-negative effect on growth and were found to be inviable with arp1Δ. The dominant-negative effects could be abrogated by either mutating the Bni1p construct to eliminate its actin-nucleating ability or adding the Cdc42-binding CRIB domain from Gic2p to enhance its localization to growth sites. Therefore the dominant-negative effects are due to inappropriate localization of actin assembly that affects both polarized growth and spindle orientation.
The results presented in this study reveal that Bni1p has four localization domains and they contribute cumulatively to Bni1p's function in polarized growth and spindle orientation. It is surprising that so many independent localization domains exist in one protein. However, multiple N-terminal localization domains seem to be a common property of yeast formin proteins, as Bnr1p has two separate localization domains, encompassing residues 1–466, and 466–733 (Gao et al., 2010 ). These findings could suggest that the cell needs very careful and intimate control of Bni1p to coordinate its localization, and hence cable assembly, with all the other events going on at sites of growth. The strict regulation of formin proteins could be common for higher organisms, as they often possess multiple formin isoforms (Higgs, 2005 ). Alternatively, the many localization domains may be used under other growth conditions, for example, during shmooing, or during the highly polarized growth that occurs during filamentous growth. The finding of four distinct localization domains implies that four different mechanisms may be involved in localizing Bni1p. Identifying these mechanisms is the next critical step in this analysis.
The strains used in this study are listed in Table 3. All strains were generated in the S288C strain background derived from the deletion consortium (Brachmann et al., 1998 ). Standard media and techniques for growing and transforming yeast were used (Sherman, 1991 ). The plasmids used in this study are listed in Table 4. Bni1p N-terminal overexpression plasmids were made using the pRS316 GAL1-10 vector pHL012 (Liu et al., 1992 ) and different Bni1p regions inserted into the BamHI and MluI restriction sites. GFP was appended using the MluI and NotI sites. To express the different Bni1p constructs, the relevant strains were first grown overnight in 5 ml SC-URA media containing 2% raffinose to an OD600 of 0.4, and then 0.5 ml of 20% galactose was added to the media and growth continued for 3–4 h. Growth assays were performed using 1:10 dilutions.
To make Bni1 internal deletion integration plasmids, 481 base pairs of the Bni1p promoter region with the first 79 amino acids of BNI1 (flanked by BamHI and MluI) were ligated with different C-terminal regions of BNI1 (flanked by MluI and NotI) and then ligated with −693 to −480 base pairs of the BNI1 promoter region (flanked by NotI and PacI) into the vector pRS305. The Bni1p internal deletion integration plasmids were then cut with NotI and transformed into appropriate strains to get the integrated BNI1 internal deletions.
To tag genomic BNI1 or BNI1 deletions with 3GFP, plasmid pWL085 was constructed using integration vector pRS306. Four DNA fragments were ligated together: BNI1 3′ UTR 6021–6171 base pairs (flanked by KpnI and SalI), the C-terminal coding region of BNI1 5500–5859 base pairs (flanked by SalI and BamHI), DNA encoding 3GFP (flanked by BamHI and NotI), and BNI1 3′ UTR 5863–6020 base pairs (flanked by NotI and SacI). This Kpn1–Sac1 fragment was then ligated into a similarly cut pRS306 vector to give pWL085. pWL085 was then digested with SalI for integration at the C-terminus of BNI1.
For live-cell imaging, cells were placed under 2% agarose in synthetic medium, with appropriate amino acids. Images were acquired on a spinning disk confocal system (3I Corp, Denver, CO) using a DMI 6000B microscope (Leica, Wetzlar, Germany) and a digital camera (QuantEM; Photometrics, Tucson, AZ). Images were then further analyzed and adjusted using SlideBook 5 (Intelligent Imaging Innovations, Denver, CO). To disrupt actin structures, cells were treated with 200 μM latrunculin A for 10 min. The actin cytoskeleton was visualized after formaldehyde fixation and staining with Alexa 568–phalloidin.
To visualize spindles, plasmid CUB1210 was linearized with XbaI and integrated into the TUB1 locus. Cells were grown at room temperature until mid-log phase, and spindle orientation was scored using GFP-Tub1p in small- to medium-budded cells. At least 100 cells were analyzed for each data point as described (Yin et al., 2000 ).
Protein extraction and immunoblotting with mouse anti-GFP monoclonal antibody (Santa Cruz Biotechnology) at 1:200 was performed as described (Santiago-Tirado et al., 2011 ).
We are very grateful to members of the Bretscher lab for comments on the manuscript. We also thank Tim Huffaker for providing plasmids that made this study possible. This work was supported by National Institutes of Health Grant GM39066.
This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E11-07-0631) on December 7, 2011.