The protein kinase kin1, the fission yeast orthologue of mammalian MARK/PAR-1, localises to new cell ends after mitosis and is important for bipolar growth

The kin1 protein kinase of the fission yeast Schizosaccharomyces pombe is a member of the PAR-1/MARK (partitioning-defective 1/microtubule-associated protein/microtubule affinity-regulating kinase) family important in eukaryotic cell polarity and cytoskeletal dynamics. We show here that kin1 plays a role in establishing the characteristic rod-shaped morphology of fission yeast. Cells in which kin1 was deleted are viable but are impaired in growth, and are rounded at one end or both ends. They are monopolar because after mitosis they fail to activate bipolar growth, and are delayed in cytokinesis, resulting in a high proportion of septated cells often with multiple septa. This phenotype can be partially rescued by heterologous expression of human MARKs, which restore bipolar growth in most cells, but do not correct the delay in cytokinesis. Using chromosomal epitope tagging, we show that kin1p localises to the cell ends, except during mitosis when it disappears from cell ends. After mitosis, kin1p first reappears at the new cell end. Overexpression of kin1 results in a loss of polarity, with partially or fully rounded cells. From these results we suggest that kin1 is required to direct the growth machinery to the cell ends.     

An overview of the KIN1/PAR-1/MARK kinase family

Members of the KIN1/PAR-1/MARK kinase family are conserved from yeast to humans and share a similar primary structural organization. Several kinases of this family appear to be at the crossroads of various biological functions including cell polarity, cell cycle control, intracellular signalisation, microtubules stability and protein stability. Here we present an overview of known roles of KIN1/PAR-1/MARK kinases including pEg3 a newly identified member which is regulated during the cell cycle and is a potential regulator of the cell cycle progression. Some common modes of action can be deciphered for this protein kinase family.     

Cell-cycle-dependent cortical localization of pEg3 protein kinase in Xenopus and human cells

BACKGROUND INFORMATION: Protein kinase pEg3 belongs to the evolutionarily conserved KIN1/PAR-1/MARK family, whose members are involved in a variety of functions, including cell polarity, microtubule stability, intracellular signalling and the cell cycle. Activity and phosphorylation of pEg3 are cell-cycle dependent and rise to maximum levels during mitosis. pEg3 was shown to interact with and phosphorylate phosphatase CDC25B, and to potentially control cell-cycle progression. Subcellular localization of pEg3 was investigated in Xenopus and human cultured cells. RESULTS: By expression of GFP (green fluorescent protein)-tagged pEg3 and indirect immunofluorescence with specific antibodies, pEg3 was found to be localized in the cytoplasm and the nucleus in interphase cells. During mitosis pEg3 was also found in the cytoplasm. From anaphase to telophase, a proportion of the protein was detected at the cell cortex. The cortical distribution in mitotic cells was dependent on F-actin, because the actin-depolymerization-inducing drugs cytochalasin D or latrunculin A prevented pEg3 cortical localization. The protein lacking the conserved C-terminal domain was not detected at the cell cortex, whereas the C-terminal domain was targeted to the cell periphery. In contrast with full-length pEg3, the cortical localization of the C-terminal domain and construct lacking the N-terminal domain was cell-cycle independent, and these constructs were found at the cell periphery in interphase cells. CONCLUSIONS: pEg3 is localized at the cell periphery specifically during mitosis. The C-terminal domain is the only pEg3 domain found to be necessary and sufficient for cortical targeting. Cortical distribution of pEg3 also requires the F-actin cytoskeleton. The cell-cycle-independent cortical localization of the pEg3 C-terminal domain and a construct lacking the N-terminal domain indicates that a negative control mechanism involving the pEg3 catalytic N-terminal domain probably acts to prevent pEg3 cortical distribution during interphase. These results suggest that pEg3 might play a role at the cell cortex during mitosis.     

Ase1p organizes antiparallel microtubule arrays during interphase and mitosis in fission yeast

Proper microtubule organization is essential for cellular processes such as organelle positioning during interphase and spindle formation during mitosis. The fission yeast Schizosaccharomyces pombe presents a good model for understanding microtubule organization. We identify fission yeast ase1p, a member of the conserved ASE1/PRC1/MAP65 family of microtubule bundling proteins, which functions in organizing the spindle midzone during mitosis. Using fluorescence live cell imaging, we show that ase1p localizes to sites of microtubule overlaps associated with microtubule organizing centers at both interphase and mitosis. ase1Delta mutants fail to form overlapping antiparallel microtubule bundles, leading to interphase nuclear positioning defects, and premature mitotic spindle collapse. FRAP analysis revealed that interphase ase1p at overlapping microtubule minus ends is highly dynamic. In contrast, mitotic ase1p at microtubule plus ends at the spindle midzone is more stable. We propose that ase1p functions to organize microtubules into overlapping antiparallel bundles both in interphase and mitosis and that ase1p may be differentially regulated through the cell cycle.     

Microtubules and actin cytoskeleton in Cryptococcus neoformans compared with ascomycetous budding and fission yeasts

Actin cytoskeleton and microtubules were studied in a human fungal pathogen, the basidiomycetous yeast Cryptococcus neoformans (haploid phase of Filobasidiella neoformans), during its asexual reproduction by budding using fluorescence and electron microscopy. Staining with rhodamine-conjugated phalloidin revealed an F-actin cytoskeleton consisting of cortical patches, cables and cytokinetic ring. F-actin patches accumulated at the regions of cell wall growth, i. e. in sterigma, bud and septum. In mother cells evenly distributed F-actin patches were joined to F-actin cables, which were directed to the growing sterigma and bud. Some F-actin cables were associated with the cell nucleus. The F-actin cytokinetic ring was located in the bud neck, where the septum originated. Antitubulin TAT1 antibody revealed a microtubular cytoskeleton consisting of cytoplasmic and spindle microtubules. In interphase cells cytoplasmic microtubules pointed to the growing sterigma and bud. As the nucleus was translocated to the bud for mitosis, the cytoplasmic microtubules disassembled and were replaced by a short intranuclear spindle. Astral microtubules then emanated from the spindle poles. Elongation of the mitotic spindle from bud to mother cell preceded nuclear division, followed by cytokinesis (septum formation in the bud neck). Electron microscopy of ultrathin sections of chemically fixed and freeze-substituted cells revealed filamentous bundles directed to the cell cortex. The bundles corresponded in width to the actin microfilament cables. At the bud neck numerous ribosomes accumulated before septum synthesis. We conclude: (i) the topology of F-actin patches, cables and rings in C. neoformans resembles ascomycetous budding yeast Saccharomyces, while the arrangement of interphase and mitotic microtubules resembles ascomycetous fission yeast Schizosaccharomyces. The organization of the cytoskeleton of the mitotic nucleus, however, is characteristic of basidiomycetous yeasts. (ii) A specific feature of C. neoformans was the formation of a cylindrical sterigma, characterized by invasion of F-actin cables and microtubules, followed by accumulation of F-actin patches around its terminal region resulting in development of an isodiametrical bud.     

Cell cycle regulation of pEg3, a new Xenopus protein kinase of the KIN1/PAR-1/MARK family.

We report the characterization of pEg3, a Xenopus protein kinase related to members of the KIN1/PAR-1/MARK family. The founding members of this newly emerging kinase family were shown to be involved in the establishment of cell polarity and both microtubule dynamic and cytoskeleton organization. Sequence analyses suggest that pEg3 and related protein kinases in human, mouse, and Caenorhabditis elegans might constitute a distinct group in this family. pEg3 is encoded by a maternal mRNA, polyadenylated in unfertilized eggs and specifically deadenylated in embryos. In addition to an increase in expression, we have shown that pEg3 is phosphorylated during oocyte maturation. Phosphorylation of pEg3 is cell cycle dependent during Xenopus early embryogenesis and in synchronized cultured XL2 cells. In embryos, the kinase activity of pEg3 is correlated to its phosphorylation state and is maximum during mitosis. Using Xenopus egg extracts we demonstrated that phosphorylation occurs at least in the noncatalytic domain of the kinase, suggesting that this domain might be important for pEg3 function.     

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.     

Fission yeast mto2p regulates microtubule nucleation by the centrosomin-related protein mto1p

From an insertional mutagenesis screen, we isolated a novel gene, mto2+, involved in microtubule organization in fission yeast. mto2Delta strains are viable but exhibit defects in interphase microtubule nucleation and in formation of the postanaphase microtubule array at the end of mitosis. The mto2Delta defects represent a subset of the defects displayed by cells deleted for mto1+ (also known as mod20+ and mbo1+), a centrosomin-related protein required to recruit the gamma-tubulin complex to cytoplasmic microtubule-organizing centers (MTOCs). We show that mto2p colocalizes with mto1p at MTOCs throughout the cell cycle and that mto1p and mto2p coimmunoprecipitate from cytoplasmic extracts. In vitro studies suggest that mto2p binds directly to mto1p. In mto2Delta mutants, although some aspects of mto1p localization are perturbed, mto1p can still localize to spindle pole bodies and the cell division site and to "satellite" particles on interphase microtubules. In mto1Delta mutants, localization of mto2p to all of these MTOCs is strongly reduced or absent. We also find that in mto2Delta mutants, cytoplasmic forms of the gamma-tubulin complex are mislocalized, and the gamma-tubulin complex no longer coimmunoprecipitates with mto1p from cell extracts. These experiments establish mto2p as a major regulator of mto1p-mediated microtubule nucleation by the gamma-tubulin complex.     

pkl1+and klp2+: Two Kinesins of the Kar3 Subfamily in Fission Yeast Perform Different Functions in Both Mitosis and Meiosis

We have identified Klp2p, a new kinesin-like protein (KLP) of the KAR3 subfamily in fission yeast. The motor domain of this protein is 61% identical and 71% similar to Pkl1p, another fission yeast KAR3 protein, yet the two enzymes are different in behavior and function. Pkl1p is nuclear throughout the cell cycle, whereas Klp2p is cytoplasmic during interphase. During mitosis Klp2p enters the nucleus where it forms about six chromatin-associated dots. In metaphase-arrested cells these migrate back and forth across the nucleus. During early anaphase they segregate with the chromosomes into two sets of about three, fade, and are replaced by other dots that form on the spindle interzone. Neither klp2(+) nor pkl1(+) is essential, and the double deletion is also wild type for both vegetative and sexual reproduction. Each deletion rescues different alleles of cut7(ts), a KLP that contributes to spindle formation and elongation. When either or both deletions are combined with a dynein deletion, vegetative growth is normal, but sexual reproduction fails: klp2 Delta,dhc1-d1 in karyogamy, pkl1 Delta,dhc1-d1 in multiple phases of meiosis, and the triple deletion in both. Deletion of Klp2p elongates a metaphase-arrested spindle, but pkl1 Delta shortens it. The anaphase spindle of klp2 Delta becomes longer than the cell, leading it to curl around the cell's ends. Apparently, Klp2p promotes spindle disassembly and contributes to the behavior of mitotic chromosomes.     

Nuclear and division-plane positioning revealed by optical micromanipulation

The position of the division plane affects cell shape and size, as well as tissue organization. Cells of the fission yeast Schizosaccharomyces pombe have a centrally placed nucleus and divide by fission at the cell center. Microtubules (MTs) are required for the central position of the nucleus. Genetic studies lead to the hypothesis that the position of the nucleus may determine the position of the division plane. Alternatively, the division plane may be positioned by the spindle or by morphogen gradients or reaction diffusion mechanisms. Here, we investigate the role of MTs in nuclear positioning and the role of the nucleus in division-plane positioning by displacing the nucleus with optical tweezers. A displaced nucleus returned to the cell center by MT pushing against the cell tips. Nuclear displacement during interphase or early prophase resulted in asymmetric cell division, whereas displacement during prometaphase resulted in symmetric division as in unmanipulated cells. These results suggest that the division plane is specified by the predividing nucleus. Because the yeast nucleus is centered by MTs during interphase but not in mitosis, we hypothesize that the establishment of the division plane at the beginning of mitosis is an optimal mechanism for accurate symmetric division in these cells.     

Importance of a myosin II-containing progenitor for actomyosin ring assembly in fission yeast

An actomyosin-based contractile ring provides the forces necessary for cell cleavage in several organisms [1-3]. Myosin II is an essential component of the actomyosin ring and has also been detected as a "spot" in interphase Schizosaccharomyces pombe cells [4-5]. It is currently unknown if this myosin II-containing spot is important for cytokinesis. In this study, we characterize this myosin II-containing spot using a combination of genetic and cell biological analyses. Whereas myosin II at the actomyosin ring undergoes rapid turnover, myosin II at the spot does not. Maintenance of the myosin II-containing spot is independent of F-actin function. Interestingly, maintenance of this myosin II spot in interphase requires the function of Rng3p, a UCS domain-containing protein, the Caenorhabditis elegans homolog of which has recently been shown to be a cochaperone for myosin II assembly [6]. Disassembly of the spot in interphase prevents actomyosin ring formation in the subsequent mitosis, implying that the spot might represent a progenitor that is important for assembly of the actomyosin ring. Given that mitosis represents a short period of the fission yeast cell cycle, organization of this progenitor structure in interphase might ensure proper assembly of the actomyosin ring and successful cell division.     

Role of the protein kinase Kin1 and nuclear centering in actomyosin ring formation in fission yeast

Cytokinesis is the last step of the cell cycle, producing two daughter cells inheriting equal genetic information. This process involves the assembly of an actomyosin ring during mitosis. In the fission yeast Schizosaccharomyces pombe, cytokinesis occurs at the geometric cell centre, a position which is defined by the interphase nucleus and the anilin-related Mid1 protein. The pom1Δ, tea1Δ and tea4Δ mutants are defective in restricting Mid1 as a band around the nucleus and misplace the division site. We previously reported that inhibition of the protein kinase Kin1 promoted failure of cytokinesis in pom1Δ and tea1Δ cells but the mechanism involving Kin1 remained elusive. Here we investigated the contribution of Kin1 in cytokinesis. We show that Kin1-GFP has a dynamic cell-cycle regulated distribution. Like pom1Δ and tea1Δ, tea4Δ exhibits a strong genetic interaction with kin1Δ. Using a conditional repressible kin1 allele that only alters interphase nuclear centering, we observed that Kin1 down-regulation severely compromised actomyosin ring formation and septum synthesis in tea4Δ cells. In addition, nuclear displacement induced either by overexpression of a putative catalytically inactive Kin1 mutant, by chemically mediated microtubule depolymerization or by mutation in the par1Δ gene impaired cytokinesis in tea4Δ but not tea4+ cells. We propose that nuclear mispositioning exacerbates the tea4Δ, pom1Δ and tea1Δ cell division phenotype. Our work reveal that nuclear centering becomes essential when Pom1/Tea1/Tea4 function is compromised and that Kin1 expression level is a key regulatory element in this situation. Our results suggest the existence of distinct overlapping control mechanisms to ensure efficient cell division.     

Self-organization of microtubule bundles in anucleate fission yeast cells

Self-organization of cellular structures is an emerging principle underlying cellular architecture. Properties of dynamic microtubules and microtubule-binding proteins contribute to the self-assembly of structures such as microtubule asters. In the fission yeast Schizosaccharomyces pombe, longitudinal arrays of cytoplasmic microtubule bundles regulate cell polarity and nuclear positioning. These bundles are thought to be organized from the nucleus at multiple interphase microtubule organizing centres (iMTOCs). Here, we find that microtubule bundles assemble even in cells that lack a nucleus. These bundles have normal organization, dynamics and orientation, and exhibit anti-parallel overlaps in the middle of the cell. The mechanisms that are responsible for formation of these microtubule bundles include cytoplasmic microtubule nucleation, microtubule release from the equatorial MTOC (eMTOC), and the dynamic fusion and splitting of microtubule bundles. Bundle formation and organization are dependent on mto1p (gamma-TUC associated protein), ase1p (PRC1), klp2p (kinesin-14) and tip1p (CLIP-170). Positioning of nuclear fragments and polarity factors by these microtubules illustrates how self-organization of these bundles contributes to establishing global spatial order.     

Small GTPase Rho5 is a functional homologue of Rho1, which controls cell shape and septation in fission yeast

The small GTPase Rho1 plays an essential role in controlling the organization of the actin cytoskeleton and synthesis of the cell wall in the fission yeast Schizosaccharomyces pombe. Here we studied the role of Rho5 whose primary structure is very similar to that of Rho1. It was found that elevated expression of Rho5 was able to compensate for the lethality of cells lacking Rho1. Rho5 was localized to the ends of interphase cells and the mid-region of mitotic cells. Overexpression of Rho5 caused depolarization of F-actin patches and abnormal formation of the cell wall, as did Rho1. Although rho5(+) was not essential for maintaining the cell shape, rho1 rho5-double null cells showed more severe defects in cell viability than rho1-null cells. Thus, it is likely that Rho5 has an overlapping function with Rho1 in controlling cell growth and division in S. pombe.     

Microtubule nucleation at non-spindle pole body microtubule-organizing centers requires fission yeast centrosomin-related protein mod20p

BACKGROUND: Many types of differentiated eukaryotic cells display microtubule distributions consistent with nucleation from noncentrosomal intracellular microtubule organizing centers (MTOCs), although such structures remain poorly characterized. In fission yeast, two types of MTOCs exist in addition to the spindle pole body, the yeast centrosome equivalent. These are the equatorial MTOC, which nucleates microtubules from the cell division site at the end of mitosis, and interphase MTOCs, which nucleate microtubules from multiple sites near the cell nucleus during interphase. RESULTS: From an insertional mutagenesis screen we identified a novel gene, mod20+, which is required for microtubule nucleation from non-spindle pole body MTOCs in fission yeast. Mod20p is not required for intranuclear mitotic spindle assembly, although it is required for cytoplasmic astral microtubule growth during mitosis. Mod20p localizes to MTOCs throughout the cell cycle and is also dynamically distributed along microtubules themselves. We find that mod20p is required for the localization of components of the gamma-tubulin complex to non-spindle pole body MTOCs and physically interacts with the gamma-tubulin complex in vivo. Database searches reveal a family of eukaryotic proteins distantly related to mod20p; these are found in organisms ranging from fungi to mammals and include Drosophila centrosomin. CONCLUSIONS: Mod20p appears to act by recruiting components of the gamma-tubulin complex to non-spindle pole body MTOCs. The identification of mod20p-related proteins in higher eukaryotes suggests that this may represent a general mechanism for the organization of noncentrosomal MTOCs in eukaryotic cells.     

Protein kinase MARK/PAR-1 is required for neurite outgrowth and establishment of neuronal polarity

Protein kinases of the microtubule affinity-regulating kinase (MARK) family were originally discovered because of their ability to phosphorylate certain sites in tau protein (KXGS motifs in the repeat domain). This type of phosphorylation is enhanced in abnormal tau from Alzheimer brain tissue and causes the detachment of tau from microtubules. MARK-related kinases (PAR-1 and KIN1) occur in various organisms and are involved in establishing and maintaining cell polarity. Herein, we report the ability of MARK2 to affect the differentiation and outgrowth of cell processes from neuroblastoma and other cell models. MARK2 phosphorylates tau protein at the KXGS motifs; this results in the detachment of tau from microtubules and their destabilization. The formation of neurites in N2a cells is blocked if MARK2 is inactivated, either by transfecting a dominant negative mutant, or by MARK2 inhibitors such as hymenialdisine. Alternatively, neurites are blocked if the target KXGS motifs on tau are rendered nonphosphorylatable by point mutations. The results suggest that MARK2 contributes to the plasticity of microtubules needed for neuronal polarity and the growth of neurites.     

CLIP170-like tip1p spatially organizes microtubular dynamics in fission yeast

Rod-shaped fission yeast cells grow in a polarized manner, and unlike budding yeast, the correct positioning of the growth sites at cell ends requires interphase microtubules. Here we describe a microtubule guidance mechanism that orients microtubules in the intracellular space along the long axis of the cell, guiding them to their target region at the cell ends. This mechanism involves tip1p, a CLIP170-like protein that localizes to distal tips of cytoplasmic microtubules. In the absence of tip1p, microtubular catastrophe is no longer restricted to cell ends but occurs when microtubules reach any region of the cellular cortex. Thus, tip1p enables microtubules to discriminate different cortical regions and regulates their dynamics accordingly.     

An anillin homologue,Mid2p,acts during fission yeast cytokinesis to organize the septin ring and promote cell separation

Anillin is a conserved protein required for cell division (Field, C.M., and B.M. Alberts. 1995. J. Cell Biol. 131:165-178; Oegema, K., M.S. Savoian, T.J. Mitchison, and C.M. Field. 2000. J. Cell Biol. 150:539-552). One fission yeast homologue of anillin, Mid1p, is necessary for the proper placement of the division site within the cell (Chang, F., A. Woollard, and P. Nurse. 1996. J. Cell Sci. 109(Pt 1):131-142; Sohrmann, M., C. Fankhauser, C. Brodbeck, and V. Simanis. 1996. Genes Dev. 10:2707-2719). Here, we identify and characterize a second fission yeast anillin homologue, Mid2p, which is not orthologous with Mid1p. Mid2p localizes as a single ring in the middle of the cell after anaphase in a septin- and actin-dependent manner and splits into two rings during septation. Mid2p colocalizes with septins, and mid2 Delta cells display disorganized, diffuse septin rings and a cell separation defect similar to septin deletion strains. mid2 gene expression and protein levels fluctuate during the cell cycle in a sep1- and Skp1/Cdc53/F-box (SCF)-dependent manner, respectively, implying that Mid2p activity must be carefully regulated. Overproduction of Mid2p depolarizes cell growth and affects the organization of both the septin and actin cytoskeletons. In the presence of a nondegradable Mid2p fragment, the septin ring is stabilized and cell cycle progression is delayed. These results suggest that Mid2p influences septin ring organization at the site of cell division and its turnover might normally be required to permit septin ring disassembly.     

Stg 1 is a novel SM22/transgelin-like actin-modulating protein in fission yeast

We identified a novel actin-modulating protein Stg 1 in the fission yeast Schizosaccharomyces pombe. Stg 1 is similar to mammalian SM22/transgelin, and biochemical experiments showed that Stg 1 crosslinked F-actin. Microscopic observation suggested that Stg 1 was a component of actin patch. Overexpression of Stg 1 caused a defect in cytokinesis by suppressing the formation of a contractile ring and formation of abnormal aggregates of F-actin in the ends and mid-region of cells. Although distribution of the actin cytoskeleton was not affected by disrupting Stg 1(+), genetic interaction suggested that Stg 1 was likely involved in controlling the organization of the actin cytoskeleton in cell morphogenesis and cytokinesis in fission yeast.