Regulation of a formin complex by the microtubule plus end protein tea1p

The plus ends of microtubules have been speculated to regulate the actin cytoskeleton for the proper positioning of sites of cell polarization and cytokinesis. In the fission yeast Schizosaccharomyces pombe, interphase microtubules and the kelch repeat protein tea1p regulate polarized cell growth. Here, we show that tea1p is directly deposited at cell tips by microtubule plus ends. Tea1p associates in large "polarisome" complexes with bud6p and for3p, a formin that assembles actin cables. Tea1p also interacts in a separate complex with the CLIP-170 protein tip1p, a microtubule plus end-binding protein that anchors tea1p to the microtubule plus end. Localization experiments suggest that tea1p and bud6p regulate formin distribution and actin cable assembly. Although single mutants still polarize, for3Deltabud6Deltatea1Delta triple-mutant cells lack polarity, indicating that these proteins contribute overlapping functions in cell polarization. Thus, these experiments begin to elucidate how microtubules contribute to the proper spatial regulation of actin assembly and polarized cell growth.     

Role of bud6p and tea1p in the interaction between actin and microtubules for the establishment of cell polarity in fission yeast

BACKGROUND: In many cell types, microtubules are thought to direct the spatial distribution of F-actin in cell polarity. Schizosaccharomyces pombe cells exhibit a regulated program of polarized cell growth: after cell division, they grow first in a monopolar manner at the old end, and in G2 phase, initiate growth at the previous cell division site (the new end). The role of microtubule ends in cell polarity is highlighted by the finding that the cell polarity factor, tea1p, is present on microtubule plus ends and cell tips [1]. RESULTS: Here, we characterize S. pombe bud6p/fat1p, a homolog of S. cerevisiae Bud6/Aip3. bud6Delta mutant cells have a specific defect in the efficient initiation of growth at the new end and like tea1Delta cells, form T-shaped cells in a cdc11 background. Bud6-GFP localizes to both cell tips and the cytokinesis ring. Maintenance of cell tip localization is dependent upon actin but not microtubules. Bud6-GFP localization is tea1p dependent, and tea1p localization is not bud6p dependent. tea1Delta and bud6Delta cells generally grow in a monopolar manner but exhibit different growth patterns. tea1(Delta)bud6Delta mutants resemble tea1Delta mutants. Tea1p and bud6p coimmunoprecipitate and comigrate in large complexes. CONCLUSIONS: Our studies show that tea1p (a microtubule end-associated factor) and bud6p (an actin-associated factor) function in a common pathway, with bud6p downstream of tea1p. To our knowledge, bud6p is the first protein shown to interact physically with tea1p. These studies delineate a pathway for how microtubule plus ends function to polarize the actin cytoskeleton through actin-associated polarity factors.     

Fission yeast Aip3p (spAip3p) is required for an alternative actin-directed polarity program

Aip3p is an actin-interacting protein that regulates cell polarity in budding yeast. The Schizosaccharomyces pombe-sequencing project recently led to the identification of a homologue of Aip3p that we have named spAip3p. Our results confirm that spAip3p is a true functional homologue of Aip3p. When expressed in budding yeast, spAip3p localizes similarly to Aip3p during the cell cycle and complements the cell polarity defects of an aip3Delta strain. Two-hybrid analysis shows that spAip3p interacts with actin similarly to Aip3p. In fission yeast, spAip3p localizes to both cell ends during interphase and later organizes into two rings at the site of cytokinesis. spAip3p localization to cell ends is dependent on microtubule cytoskeleton, its localization to the cell middle is dependent on actin cytoskeleton, and both patterns of localization require an operative secretory pathway. Overexpression of spAip3p disrupts the actin cytoskeleton and cell polarity, leading to morphologically aberrant cells. Fission yeast, which normally rely on the microtubule cytoskeleton to establish their polarity axis, can use the actin cytoskeleton in the absence of microtubule function to establish a new polarity axis, leading to the formation of branched cells. spAip3p localizes to, and is required for, branch formation, confirming its role in actin-directed polarized cell growth in both Schizosaccharomyces pombe and Saccharomyces cerevisiae.     

The protein kinase kin1 is required for cellular symmetry in fission yeast

The fission yeast Schizosaccharomyces pombe is a highly polarized unicellular eukaryote with two opposite growing poles in which F-actin cytoskeleton is focused. The KIN1/PAR-1/MARK protein family is composed of conserved eukaryotic serine/threonine kinases which are involved in cell polarity, microtubule stability or cell cycle regulation. Here, we investigate the function of the fission yeast KIN1/PAR-1/MARK member, kin1p. Using a deletion allele (kin1Delta), we show that kin1 mutation promotes a delay in septation. Kin1p regulates the structure of the new cell end after cytokinesis by modulating cell wall remodeling. Abnormal shaped interphase kin1Delta cells misplace F-actin patches and the premitotic nucleus. Thus, mitotic kin1Delta cells misposition the F-actin ring assembly site that is dependent on the position of the interphase nucleus. The resulting asymmetric cell division produces daughter cells with distinct shapes. Overexpressed kin1p accumulates asymmetrically at the cell cortex and affects cell shape, F-actin organization and microtubules. Our results suggest that correct dosage of kin1p at the cortex is required for spatial organization of the fission yeast cell.     

Function of rax2p in the polarized growth of fission yeast

Cell polarity is critical for the division, differentiation, migration, and signaling of eukaryotic cells. RAX2 of budding yeast encodes a membrane protein localized at the cell cortex that helps maintain the polarity of the bipolar pattern. Here, we designate SPAC6f6.06c as rax2+ of Schizosaccharomyces pombe, based on its sequence homology with RAX2, and examine its function in cell polarity. S. pombe rax2+ is not essential, but Deltarax2 cells are slightly smaller and grow slower than wild type cells. During vegetative growth or arrest at G1 by mutation of cdc10, deletion of rax2+ increases the number of cells failing old end growth just after division. In addition, this failure of old end growth is dramatically increased in Deltatea1Deltarax2, pointing to genetic interaction of rax2+ with tea1+. Deltarax2 cells contain normal actin and microtubule cytoskeletons, but lack actin cables, and the polarity factor for3p is not properly localized at the growing tip. In Deltarax2 cells, and endogenous rax2p is localized at the cell cortex of growing cell tips in an actin- and microtubule-dependent manner. However, Deltarax2 cells show no defects in cell polarity during shmoo formation and conjugation. Taken together, these observations suggest that rax2p controls the cell polarity of fission yeast during vegetative growth by regulating for3p localization.     

Movement of a cytokinesis factor cdc12p to the site of cell division.

A key question in cytokinesis is how the plane of cell division is positioned within the cell. Although a number of cytokinesis factors involved in formation of the actomyosin contractile ring have been identified, little is known about how these factors are localized and assembled at the cell-division site. Cells of the fission yeast Schizosaccharomyces pombe divide using a medial actomyosin ring that assembles in early mitosis [1]. The S. pombe cdc12 gene encodes a formin, a member of a family of proteins that have functions in cytokinesis and cell polarity and that may bind Rho/Cdc42 GTPases, profilin and other actin-associated proteins [1] [2] [3] [4]. The cdc12 protein (cdc12p) is required specifically for medial-ring assembly during cytokinesis and is a component of this ring [2] [5]. In this study, cdc12p was found, during interphase, in a discrete, motile cytoplasmic spot that moved to the future site of cell division at the onset of mitosis. Three lines of evidence indicated that this cdc12p spot moved on both actin and microtubule networks: movement required either actin or microtubules; the spot was associated with actin and microtubule structures; and individual spots were seen to move along both microtubule and non-microtubule tracks. These findings demonstrate that a cytokinesis factor may travel on both microtubule and actin networks to the future site of cell division.     

The Saccharomyces cerevisiae Arf3 protein is involved in actin cable and cortical patch formation

We show that Arf3p, a member of the ADP ribosylation family, is involved in the organization of actin cables and cortical patches in Saccharomyces cerevisiae. Profilin-deficient cells (pfy1Delta) have severe growth defects and lack actin cables. Overexpression of ARF3 restores actin cables and corrects growth defects in these cells. Cells deficient for the cortical patch proteins Las17p and Vrp1p have growth defects and a random cortical patch distribution. Overexpression of ARF3 in las17Delta and in vrp1Delta cells partially corrects growth defects and restores the polarized distribution of cortical patches. The N-terminal glycine, a myristoylation site in Arf3p, is necessary for its suppressor activity. arf3Delta cells show a random budding pattern. Overexpression of BNI1, GEA2 or SYP1, three genes involved in actin cytoskeleton formation, restores the normal axial budding pattern of arf3Delta cells. BUD6 is a polarity gene and GEA2 is involved in retrograde transport and the organization of the actin cytoskeleton. We have identified genetic interactions between ARF3 and BUD6, and between ARF3 and GEA2. Both double mutant strains have actin cytoskeleton defects. Our results support a role for ARF3 in cell polarity and the organization of the actin cytoskeleton.     

discordia mutations specifically misorient asymmetric cell divisions during development of the maize leaf epidermis.

In plant cells, cytokinesis depends on a cytoskeletal structure called a phragmoplast, which directs the formation of a new cell wall between daughter nuclei after mitosis. The orientation of cell division depends on guidance of the phragmoplast during cytokinesis to a cortical site marked throughout prophase by another cytoskeletal structure called a preprophase band. Asymmetrically dividing cells become polarized and form asymmetric preprophase bands prior to mitosis; phragmoplasts are subsequently guided to these asymmetric cortical sites to form daughter cells of different shapes and/or sizes. Here we describe two new recessive mutations, discordia1 (dcd1) and discordia2 (dcd2), which disrupt the spatial regulation of cytokinesis during asymmetric cell divisions. Both mutations disrupt four classes of asymmetric cell divisions during the development of the maize leaf epidermis, without affecting the symmetric divisions through which most epidermal cells arise. The effects of dcd mutations on asymmetric cell division can be mimicked by cytochalasin D treatment, and divisions affected by dcd1 are hypersensitive to the effects of cytochalasin D. Analysis of actin and microtubule organization in these mutants showed no effect of either mutation on cell polarity, or on formation and localization of preprophase bands and spindles. In mutant cells, phragmoplasts in asymmetrically dividing cells are structurally normal and are initiated in the correct location, but often fail to move to the position formerly occupied by the preprophase band. We propose that dcd mutations disrupt an actin-dependent process necessary for the guidance of phragmoplasts during cytokinesis in asymmetrically dividing cells.     

Establishment of a cellular axis in fission yeast

Recent studies in fission yeast Schizosaccharomyces pombe reveal how cells establish a cellular axis that specifies domains as the functional 'ends' and 'middle' of the cell. During interphase, dynamic microtubules position the nucleus at the middle of the cell and orientate microtubule 'plus' ends towards the ends of the cell. At the cell ends, the microtubule plus ends might establish a zone of polarized cell growth and actin assembly by depositing factors such as Tea1p. At the cell middle, the nucleus might specify the position of the actin contractile ring and the future cell division site by positioning cytokinesis factors such as Mid1p.     

New End Take Off: Regulating Cell Polarity during the Fission

Cell polarisation is a major event of the cell cycle and underlies the function of mostcells. Cell polarity is often achieved through the coordinated organisation of themicrotubule and actin cytoskeletons. Dramatic changes in cell polarisation occur duringthe cell cycle and are subject to regulation by cell cycle controls. Cells of the fission yeastSchizosaccharomyces pombe grow by tip extension in a cell cycle-controlled manner.During G2 phase, these cells exhibit a transition in cell polarisation known as New EndTake Off (NETO), in which monopolar cells initiate bipolar growth. Dynamicmicrotubules contribute to this process by depositing at cell ends the microtubule plusend proteins tea1p and tea4p, which are necessary for NETO. We discuss here how theseproteins may recruit for3p, a formin responsible for actin nucleation, as well as two otheractin binding proteins, bud6p and sla2p, to initiate cell polarisation at the new end of thecell. Thus, the study of NETO is revealing a mechanism by which the plus ends ofmicrotubules regulate the spatial organisation of actin.     

Opposing roles for actin in Cdc42p polarization

In animal and fungal cells, the monomeric GTPase Cdc42p is a key regulator of cell polarity that itself exhibits a polarized distribution in asymmetric cells. Previous work showed that in budding yeast, Cdc42p polarization is unaffected by depolymerization of the actin cytoskeleton (Ayscough et al., J. Cell Biol. 137, 399-416, 1997). Surprisingly, we now report that unlike complete actin depolymerization, partial actin depolymerization leads to the dispersal of Cdc42p from the polarization site in unbudded cells. We provide evidence that dispersal is due to endocytosis associated with cortical actin patches and that actin cables are required to counteract the dispersal and maintain Cdc42p polarity. Thus, although Cdc42p is initially polarized in an actin-independent manner, maintaining that polarity may involve a reinforcing feedback between Cdc42p and polarized actin cables to counteract the dispersing effects of actin-dependent endocytosis. In addition, we report that once a bud has formed, polarized Cdc42p becomes more resistant to dispersal, revealing an unexpected difference between unbudded and budded cells in the organization of the polarization site.     

Actin-mediated Delivery of Astral Microtubules Instructs Kar9p Asymmetric Loading to the Bud-Ward Spindle Pole

In Saccharomyces cerevisiae, Kar9p, one player in spindle alignment, guides the bud-ward spindle pole by linking astral microtubule plus ends to Myo2p-based transport along actin cables generated by the formins Bni1p and Bnr1p and the polarity determinant Bud6p. Initially, Kar9p labels both poles but progressively singles out the bud-ward pole. Here, we show that this polarization requires cell polarity determinants, actin cables, and microtubules. Indeed, in a bud6Δ bni1Δ mutant or upon direct depolymerization of actin cables Kar9p symmetry increased. Furthermore, symmetry was selectively induced by myo2 alleles, preventing Kar9p binding to the Myo2p cargo domain. Kar9p polarity was rebuilt after transient disruption of microtubules, dependent on cell polarity and actin cables. Symmetry breaking also occurred after transient depolymerization of actin cables, with Kar9p increasing at the spindle pole engaging in repeated cycles of Kar9p-mediated transport. Kar9p returning to the spindle pole on shrinking astral microtubules may contribute toward this bias. Thus, Myo2p transport along actin cables may support a feedback loop by which delivery of astral microtubule plus ends sustains Kar9p polarized recruitment to the bud-ward spindle pole. Our findings also explain the link between Kar9p polarity and the choice setting aside the old spindle pole for daughter-bound fate.     

Mto2p, a novel fission yeast protein required for cytoplasmic microtubule organization and anchoring of the cytokinetic actin ring

Microtubules regulate diverse cellular processes, including chromosome segregation, nuclear positioning, and cytokinesis. In many organisms, microtubule nucleation requires gamma-tubulin and associated proteins present at specific microtubule organizing centers (MTOCs). In fission yeast, interphase cytoplasmic microtubules originate from poorly characterized interphase MTOCs and spindle pole body (SPB), and during late anaphase from the equatorial MTOC (EMTOC). It has been previously shown that Mto1p (Mbo1p/Mod20p) function is important for the organization/nucleation of all cytoplasmic microtubules. Here, we show that Mto2p, a novel protein, interacts with Mto1p and is important for establishing a normal interphase cytoplasmic microtubule array. In addition, mto2Delta cells fail to establish a stable EMTOC and localize gamma-tubulin complex members to this medial structure. As predicted from these functions, Mto2p localizes to microtubules, the SPB, and the EMTOC in an Mto1p-dependent manner. mto2Delta cells fail to anchor the cytokinetic actin ring in the medial region of the cell and under conditions that mildly perturb actin structures, these rings unravel in mto2Delta cells. Our results suggest that the Mto2p and the EMTOC are critical for anchoring the cytokinetic actin ring to the medial region of the cell and for proper coordination of mitosis with cytokinesis.     

Tea4p links microtubule plus ends with the formin for3p in the establishment of cell polarity

Microtubules regulate actin-based processes such as cell migration and cytokinesis, but molecular mechanisms are not understood. In the fission yeast Schizosaccharomyces pombe, microtubule plus ends regulate cell polarity in part by transporting the kelch repeat protein tea1p to cell ends. Here, we identify tea4p, a SH3 domain protein that binds directly to tea1p. Like tea1p, tea4p localizes to growing microtubule plus ends and to cortical sites at cell ends, and it is necessary for the establishment of bipolar growth. Tea4p binds directly to and recruits the formin for3p, which nucleates actin cable assembly. During "new end take off" (NETO), formation of a protein complex that includes tea1p, tea4p, and for3p is necessary and sufficient for the establishment of cell polarity and localized actin assembly at new cell ends. Our results suggest a molecular mechanism for how microtubule plus ends regulate the spatial distribution of actin assembly.     

Pob1 ensures cylindrical cell shape by coupling two distinct rho signaling events during secretory vesicle targeting

Proper cell morphogenesis requires the co-ordination of cell polarity, cytoskeletal organization and vesicle trafficking. The Schizosaccharomyces pombe mutant pob1-664 has a curious lemon-like shape, the basis of which is not understood. Here, we found abundant vesicle accumulation in these cells, suggesting that Pob1 plays a role in vesicle trafficking. We identified Rho3 as a multicopy suppressor of this phenotype. Because Rho3 function is related to For3, an actin-polymerizing protein, and Sec8, a component of the exocyst complex, we analyzed their functional relationship with Pob1. Pob1 was essential for the formation of actin cables (by interacting with For3) and for the polarized localization of Sec8. Although neither For3 nor Sec8 is essential for polarized growth, their simultaneous disruption prevented tip growth and yielded a lemon-like cell morphology similar to pob1-664. Thus, Pob1 may ensure cylindrical cell shape of S. pombe by coupling actin-mediated vesicle transport and exocyst-mediated vesicle tethering during secretory vesicle targeting.     

Tea2p is a kinesin-like protein required to generate polarized growth in fission yeast

Cytoplasmic microtubules are critical for establishing and maintaining cell shape and polarity. Our investigations of kinesin-like proteins (klps) and morphological mutants in the fission yeast Schizosaccharomyces pombe have identified a kinesin-like gene, tea2(+), that is required for cells to generate proper polarized growth. Cells deleted for this gene are often bent during exponential growth and initiate growth from improper sites as they exit stationary phase. They have a reduced cytoplasmic microtubule network and display severe morphological defects in genetic backgrounds that produce long cells. The tip-specific marker, Tea1p, is mislocalized in both tea2-1 and tea2Delta cells, indicating that Tea2p function is necessary for proper localization of Tea1p. Tea2p is localized to the tips of the cell and in a punctate pattern within the cell, often coincident with the ends of cytoplasmic microtubules. These results suggest that this kinesin promotes microtubule growth, possibly through interactions with the microtubule end, and that it is important for establishing and maintaining polarized growth along the long axis of the cell.     

Fission yeast Pom1p kinase activity is cell cycle regulated and essential for cellular symmetry during growth and division

Schizosaccharomyces pombe cells grow from both ends during most of interphase and divide symmetrically into two daughter cells. The pom1 gene, encoding a member of the Dyrk family of protein kinases, has been identified through a mutant showing abnormal cellular morphogenesis. Here we show that Pom1p kinase activity is cell cycle regulated in correlation with the state of cellular symmetry: the activity is high during symmetrical growth and division, but lower when cells grow at just one end. Point mutations in the catalytic domain lead to asymmetry during both cell growth and division, whilst cells overexpressing Pom1p form additional growing ends. Manipulations of kinase activity indicate a negative role for Pom1p in microtubule growth at cell ends. Pom1p is present in a large protein complex and requires its non-catalytic domain to localize to the cell periphery and its kinase activity to localize to cell ends. These data establish that Pom1p kinase activity plays an important role in generating cellular symmetry and suggest that there may be related roles of homologous protein kinases ubiquitously present in all eukaryotes.     

Cell polarity from cell division

Apical-basal polarity of epithelial cells is critical for their symmetric versus asymmetric division and commonly thought to be established in interphase. In a novel type of cell division termed "mirror-symmetric", apical cell constituents accumulate during M-phase at the cleavage furrow, resulting in epithelial daughter cells with opposite apical-basal polarity.     

The secretory pathway mediates localization of the cell polarity regulator Aip3p/Bud6p

Aip3p/Bud6p is a regulator of cell and cytoskeletal polarity in Saccharomyces cerevisiae that was previously identified as an actin-interacting protein. Actin-interacting protein 3 (Aip3p) localizes at the cell cortex where cytoskeleton assembly must be achieved to execute polarized cell growth, and deletion of AIP3 causes gross defects in cell and cytoskeletal polarity. We have discovered that Aip3p localization is mediated by the secretory pathway. Mutations in early- or late-acting components of the secretory apparatus lead to Aip3p mislocalization. Biochemical data show that a pool of Aip3p is associated with post-Golgi secretory vesicles. An investigation of the sequences within Aip3p necessary for Aip3p localization has identified a sequence within the N terminus of Aip3p that is sufficient for directing Aip3p localization. Replacement of the N terminus of Aip3p with a homologous region from a Schizosaccharomyces pombe protein allows for normal Aip3p localization, indicating that the secretory pathway-mediated Aip3p localization pathway is conserved. Delivery of Aip3p also requires the type V myosin motor Myo2p and its regulatory light-chain calmodulin. These data suggest that one function of calmodulin is to activate Myo2p's activity in the secretory pathway; this function is likely the polarized movement of late secretory vesicles and associated Aip3p on actin cables.