Saccharomyces cerevisiae Kelch Proteins and Bud14 Protein Form a Stable 520-kDa Formin Regulatory Complex That Controls Actin Cable Assembly and Cell Morphogenesis

Formins perform essential roles in actin assembly and organization in vivo, but they also require tight regulation of their activities to produce properly functioning actin structures. Saccharomyces cerevisiae Bud14 is one member of an emerging class of formin regulators that target the FH2 domain to inhibit actin polymerization, but little is known about how these regulators are themselves controlled in vivo. Kelch proteins are critical for cell polarity and morphogenesis in a wide range of organisms, but their mechanistic roles in these processes are still largely undefined. Here, we report that S. cerevisiae Kelch proteins, Kel1 and Kel2, associate with Bud14 in cell extracts to form a stable 520-kDa complex with an apparent stoichiometry of 2:2:1 Bud14/Kel1/Kel2. Using pairwise combinations of GFP- and red fluorescent protein-tagged proteins, we show that Kel1, Kel2, and Bud14 interdependently co-localize at polarity sites. By analyzing single, double, and triple mutants, we show that Kel1 and Kel2 function in the same pathway as Bud14 in regulating Bnr1-mediated actin cable formation. Loss of any component of the complex results in long, bent, and hyper-stable actin cables, accompanied by defects in secretory vesicle traffic during polarized growth and septum formation during cytokinesis. These observations directly link S. cerevisiae Kelch proteins to the control of formin activity, and together with previous observations made for S. pombe homologues tea1p and tea3p, they have broad implications for understanding Kelch function in other systems.     

Closing the loops: new insights into the role and regulation of actin during cell polarization

In this review, we summarize recent results on the understanding of actin organization and cell polarization with an emphasis on the critical role of actin during this process. We first report on the advances made in understanding the function and mechanism of formin family proteins in the nucleation of actin filaments. We also discuss how formins and other regulators of actin dynamics are thought to be involved in the generation of cell polarity. In the second part we discuss new findings indicating that, rather than using a linear pathway from signal transduction to cytoskeleton re-organization, cell polarity is established through bidirectional interplay between these processes. We describe the various types of feedback loops identified and point out common schemes. Finally we briefly summarize the emerging role of actinlike proteins in the generation of polarity in prokaryotes that implies an early origin of actin's role in cell polarity.     

Shape and size changes induced by taurine depletion in neonatal cardiomyocytes

Summary Taurine is a very important organic osmolyte in most adult cells. Because of this property it has been proposed that large changes in the intracellular content of taurine can osmotically stress the cell, causing changes in its size and shape. This hypothesis was examined by measuring cell dimensions of taurine deficient cardiomyocytes using confocal microscopy. Incubation of isolated neonatal rat myocytes with medium containing 5mM-alanine led to a 55% decrease in intracellular taurine content. Associated with the loss of taurine was a reduction in cell size. Two factors contributed to the change in cell size. First, there was a shift in cell shape, favoring the smaller of the two cellular configurations commonly found in the myocyte cell culture. Second, the size of the polyhedral configuration was reduced after ßalanine treatment. These same two events also contributed to size reduction in cardiomyocytes incubated with medium containing 30mM mannitol. Nonetheless, some qualitative differences exist between cells osmotically stressed by increasing the osmolality of the incubation medium and decreasing intracellular osmolality. The results support a role for taurine in the regulation of osmotic balance in the neonatal cardiomyocyte.     

细胞壁在细胞极性建立和胚胎发生中的作用

植物细胞壁是一个活性的动态结构,其结构层次与组分随着发育进程而发生变化,且广泛参与细胞的各项生命活动,特别是在参与细胞命运决定、充当细胞发育信使、调控植物胚胎早期极性建立以及模式建成等方面发挥重要作用。     

Cytokinetic abscission in animal cells

Cytokinesis leads to the separation of dividing cells, which in animal cells involves the contraction of an actin–myosin ring and subsequent fission during abscission. Abscission requires a series of dynamic events, including midbody-targeted vesicle secretion, specialization of plasma membrane domains, disassembly of midbody-associated microtubule bundles and plasma membrane fission. A large number of molecular factors required for abscission have been identified through localization, loss-of-function and proteomics studies, but their coordinate function in abscission is still poorly understood. Here, we review the structural elements and molecular factors known to contribute to abscission, and discuss their potential role in the context of proposed models for the abscission mechanism.     

EphrinA1-EphA2 signal induces compaction and polarization of Madin-Darby canine kidney cells by inactivating Ezrin through negative regulation of RhoA

The epithelial cells exhibit either a columnar or a flat shape dependent on extracellular stimuli or the cell-cell adhesion. Membrane-anchored ephrinA stimulates EphA receptor tyrosine kinases as a ligand in a cell-cell contact-dependent manner. The mechanism through which ephrinA1/EphA2 signal regulates the cell morphology remains elusive. We demonstrate here that ephrinA1/EphA2 signal induces compaction and enhanced polarization (columnar change) of Madin-Darby canine kidney epithelial cells by regulating Ezrin, a linker that connects plasma membrane and actin cytoskeleton. Activation of EphA2 resulted in RhoA inactivation through p190RhoGAP-A and subsequent dephosphorylation of Ezrin on Thr-567 phosphorylated by Rho kinase. Consistently, the cells expressing an active mutant of Ezrin in which Thr-567 was replaced with Asp did not change their shape in response to ephrinA1. Furthermore, depletion of Ezrin led to compaction and enhanced polarization without ephrinA1 stimulation, suggesting the role for active Ezrin in keeping the flat cell shape. Ezrin localized to apical domain irrespective of ephrinA1 stimulation, whereas phosphorylated Ezrin on the apical domain was reduced by ephrinA1 stimulation. Collectively, ephrinA1/EphA2 signal negatively regulates Ezrin and promotes the alteration of cell shape, from flat to columnar shape.     

Cytokinesis: an emerging unified theory for eukaryotes?

In animal and fungal cells, cytokinesis involves an actomyosin ring that forms and contracts at the division plane. Important new details have emerged concerning the composition, assembly, and dynamics of these contractile rings. In addition, recent advances suggest that targeted membrane addition is a central feature of cytokinesis in animal cells - as it is in fungi and plants - and the coordination of actomyosin ring function with targeted exocytosis at the cleavage plane is being explored. Important new information has also emerged about the spatial and temporal regulation of cytokinesis, especially in relation to the function of the spindle midzone in animal cells and the control of cytokinesis by GTPase systems.     

Induction,polarity and spatial control of cytokinesis in some abnormal subsidiary cell mother cells ofZea mays

Summary Cytokinesis in the subsidiary cell mother cells (SMCs) ofZea mays leaves grown in the presence of 5 mM of caffeine solution is usually partially inhibited. A continuous wall strip, resembling a portion of the subsidiary cell (SC) wall, is laid down in the preprophase microtubule band (PMB) cortical zone. Sometimes, the incomplete SC (SC) wall grows centripetally in the absence of a phragmoplast and the gap becomes smaller or closes. The SC nucleus escapes through the SC wall gap into the larger SMC compartment and may fuse with the other nucleus.The aberrant SMCs (a-SMCs) pass through another division cycle, reattempting to produce a SC. A typical PMB is found in the SC space, in the site of the previous PMB. Moreover, in some preprophase SMCs, the cytoplasm adjacent to the SC wall is traversed by a small number of microtubules. The preprophase nuclei are partly or totally separated from the PMB by the perforated SC wall and may lie far from the latter.Usually, one mitotic spindle is assembled. The cycling paired polarized nuclei appear to synchronize and their chromosomes line up together on a single metaphase plate. Although the mitotic spindle axis is diversely oriented, one of its poles tends to be stabilized in the proximity of the SC wall gap. These divisions separate abnormal cells. Most or all the cell plate edges fuse with wall regions far from the PMB cortical zone. However, when some of them approach the SC wall strips, they are attracted and intersect their rims. In rare occasions the cell plate, invading the SC space is guided by the PMB cortical zone to create a SC-like curved wall portion, in absence of a daughter nucleus.Observations show that the cell plate arrangement in redividing aberrant SMCs is not subjected to a strict spatial control. The disorder of polarization sequence generated by the SC wall ring and especially the perturbation of the spatial (and functional?) relationship between PMB-PMB cortical zone and the nucleus—mitotic spindle is a causal factor of the variable cell plate arrangements.     

SEC14 and Spectrin Domains 1 (Sestd1) and Dapper Antagonist of Catenin 1 (Dact1) Scaffold Proteins Cooperatively Regulate the Van Gogh-like 2 (Vangl2) Four-pass Transmembrane Protein and Planar Cell Polarity (PCP) Pathway during Embryonic Development in Mice

The planar cell polarity (PCP) pathway is a conserved non-canonical (β-catenin-independent) branch of Wnt signaling crucial to embryogenesis, during which it regulates cell polarity and polarized cell movements. Disruption of PCP components in mice, including Vangl2 and Dact1, results in defective neural tube closure and other developmental defects. Here, we show that Sestd1 is a novel binding partner of Vangl2 and Dact1. The Sestd1-Dact1 interface is formed by circumscribed regions of Sestd1 (the carboxyl-terminal region) and Dact1 (the amino-terminal region). Remarkably, we show that loss of Sestd1 precisely phenocopies loss of Dact1 during embryogenesis in mice, leading to a spectrum of birth malformations, including neural tube defects, a shortened and/or curly tail, no genital tubercle, blind-ended colons, hydronephrotic kidneys, and no bladder. Moreover, as with Dact1, a knock-out mutation at the Sestd1 locus exhibits reciprocal genetic rescue interactions during development with a semidominant mutation at the Vangl2 locus. Consistent with this, examination of Wnt pathway activities in Sestd1 mutant mouse embryonic tissue reveals disrupted PCP pathway biochemistry similar to that characterized in Dact1 mutant embryos. The Sestd1 protein is a divergent member of the Trio family of GTPase regulatory proteins that lacks a guanine nucleotide exchange factor domain. Nonetheless, in cell-based assays the Sestd1-Dact1 interaction can induce Rho GTPase activation. Together, our data indicate that Sestd1 cooperates with Dact1 in Vangl2 regulation and in the PCP pathway during mammalian embryonic development.     

银杏雄配子体发生发育过程中的细胞分裂

银杏（ＧｉｎｋｇｏｂｉｌｏｂａＬ．）小孢子母细胞减数分裂中,拟核经过规律性的变化后,初步建立了由近极面到远极面间的轴向极性,因而,雄配子体萌发时的３次分裂都是典型的极性平周分裂;这些极性平周分裂很可能是对原有极性的进一步加强;在结构上,各子细胞间的细胞壁缺少胞间连丝,因而,这些细胞壁可能起着使子细胞孤立化的作用,从而完成雄配子体中各细胞间的精细分化。生殖细胞的分裂很可能是斜背式环形分裂（ａｎｔｉｃｌｉｎａｌｒｉｎｇｌｉｋｅｄｉｖｉｓｉｏｎ）,这种分裂可能是对最初极性方向的重大调整。结果,精原细胞的分裂方向为垂周分裂,产生两个背靠背排列的精子。     

Cytokinesis and cancer

Cytokinesis is the final stage of cell division during which the two daughter cells separate completely. Although less well understood than some of the earlier phases of the cell cycle, recent discoveries have shed light on the mechanisms that orchestrate this process, including cleavage furrow formation, midbody maturation and abscission. One of the reasons why research on cytokinesis has been attracting increasing attention is the concept that failure of this process in mammals is associated with carcinogenesis. In this minireview, we will discuss the possible links between cytokinesis and cancer, and highlight key mechanisms that connect these processes.     

Cholesterol is essential for mitosis progression and its deficiency induces polyploid cell formation

As an essential component of mammalian cell membranes, cells require cholesterol for proliferation, which is either obtained from plasma lipoproteins or synthesized intracellularly from acetyl-CoA. In addition to cholesterol, other non-sterol mevalonate derivatives are necessary for DNA synthesis, such as the phosphorylated forms of isopentane, farnesol, geranylgeraniol, and dolichol. The aim of the present study was to elucidate the role of cholesterol in mitosis. For this, human leukemia cells (HL-60) were incubated in a cholesterol-free medium and treated with SKF 104976, which inhibits cholesterol biosynthesis by blocking sterol 14alpha-demethylase, and the expression of relevant cyclins in the different phases of the cell cycle was analyzed by flow cytometry. Prolonged cholesterol starvation induced the inhibition of cytokinesis and the formation of polyploid cells, which were multinucleated and had mitotic aberrations. Supplementing the medium with cholesterol completely abolished these effects, demonstrating they were specifically due to cholesterol deficiency. This is the first evidence that cholesterol is essential for mitosis completion and that, in the absence of cholesterol, the cells fail to undergo cytokinesis, entered G1 phase at higher DNA ploidy (tetraploidy), and then progressed through S (rereplication) into G2, generating polyploid cells.     

Division-site selection, cell separation, and formation of anucleate minicells inSchizosaccharomyces pombe mutants resistant to cell-wall lytic enzymes

Summary In most eukaryotic organisms that have cell walls, cell separation or cytokinesis is a degradative enzymatic process. In the fission yeastSchizosaccharomyces pombe, it is a post-M-phase event that includes the degradation of part of the cell wall and the primary septum. We describe the isolation of mutants partially defective in cytokinesis by enrichment of clones resistant to cell-wall lytic enzymes. The mutations confer mycelial morphology (chains of non-separated cells) and define four genes.Sep2-SA2 was subjected to detailed genetic and cytological analysis. Its cells frequently form complex septa composed of multiple layers, which appear as twin septa separated by anucleate minicells if the cell length is extended. This suggests that a polar signal-like mechanism may also operate inS. pombe during division-site selection andsep2 ⁺ takes part in it.Sep2 ⁺ seems to be involved in several cell cycle functions because its mutation can transiently block cell-cycle progression after nuclear division and provoke a transition from haploidy to diploidy in the double mutantsep2-SA2 cexl-SA2. Cexl-SA2 is another novel mutation which causes cell-length extension.Abbreviations DAPI 4,6-diamidino-2-phenylindole - YEA yeast extract agar - YEL yeast extract liquid - SMA synthetic minimal agar - MEA malt extract agar     

The formation and patterning of leaves: recent advances

Leaves, the plants major photosynthetic organs, form through the activity of groups of pluripotent cells, termed shoot apical meristems (SAMs), located at the growing tips of plants. Leaves develop with a dorso–ventral asymmetry, with the adaxial surface adjacent to the meristem and the abaxial surface developing at a distance from it. Molecular genetic studies have shown that the correct specification of adaxial/abaxial polarity requires communication between the incipient leaf and the meristem, and that the juxtaposition of adaxial/abaxial fates is necessary for lamina outgrowth (Waites and Hudson 1995; McConnell et al. 2001). Over the last few years, a number of factors that control cell fate specification in the apex have been identified. This review will focus on recent advances on distinct but overlapping aspects of leaf development, namely, the transition from meristem to leaf fate and the specification of abaxial/adaxial polarity and its possible role in leaf growth.     

Ultrastructure of mouse blastocysts during blastocoele expansion

Summary The ultrastructure of mouse blastocysts with nascent and expanded blastocoele is described. In the early blastocyst cells adhere tightly and the blastocoele is often limited at its apex by cells containing a midbody. The expanding blastocyst exhibits a loose cell arrangement due to the presence of intercellular spaces and a cortical layer of filaments develops in cells enclosing the expanded blastocoele. When the blastocoele exceeds 1/2 the embryo diameter desmosomes appear between trophectoderm cells. Possible factors essential for blastocoele formation are discussed.     

The human small glutamine-rich TPR-containing protein is required for progress through cell division

Eukaryotic organisms from yeast to human harbor genes encoding the small glutamine-rich tetratricopeptide repeat-containing (SGT) protein. Work presented here demonstrated the presence of human SGT (hSGT) protein in a panel of human cell lines and throughout the cell cycle. To identify cellular processes in which hSGT is involved, knock down populations were analyzed which were generated through transfection of hsgt-specific small interfering RNA. Most strikingly, depletion of hSGT led to reduced proliferation of the affected cell populations while the mitotic index was increased. Time-lapse video microscopy revealed that cells from hSGT-depleted populations were unable to complete cell division due to mitotic arrest which was frequently followed by cell death. Further evidence for a role in cell division was given by the accumulation of hSGT in the midzone and the midbody, and by a mitosis-specific migration pattern of hSGT as detected by Western blotting after SDS-PAGE or two-dimensional gel electrophoresis. In conclusion, results obtained in this study demonstrate that hSGT protein is a constitutive component of all human cell lines tested and that this protein is essential for successful completion of cell division.     

Divide and conquer: cytokinesis in plant cells

Plant cells divide in two by constructing a new cell wall (cell plate) between daughter nuclei after mitosis. Golgi-derived vesicles are transported to the equator of a cytoskeletal structure called a phragmoplast, where they fuse together to form the cell plate. Orientation of new cell walls involves actindependent guidance of phragmoplasts and associated cell plates to cortical sites established prior to mitosis. Recent work has provided new insights into how actin filaments and other proteins in the phragmoplast and cell plate contribute to cytokinesis. Newly discovered mutations have identified a variety of genes required for cytokinesis or its spatial regulation.     

Structural Basis of the Binding of Merlin FERM Domain to the E3 Ubiquitin Ligase Substrate Adaptor DCAF1

The tumor suppressor gene Nf2 product, Merlin, plays vital roles in controlling proper development of organ sizes by specifically binding to a large number of target proteins localized both in cytoplasm and nuclei. The FERM domain of Merlin is chiefly responsible for its binding to target proteins, although the molecular basis governing these interactions are poorly understood due to lack of structural information. Here, we report the crystal structure of the Merlin FERM domain in complex with its binding domain derived from the E3 ubiquitin ligase substrate adaptor DCAF1 (also known as VPRBP). Unlike target binding modes found in ERM proteins, the Merlin-FERM binding domain of DCAF1 folds as a β-hairpin and binds to the α1/β5-groove of the F3 lobe of Merlin-FERM via extensive hydrophobic interactions. In addition to providing the first structural glimpse of a Merlin-FERM·target complex, the structure of the Merlin·DCAF1 complex is likely to be valuable for understanding the interactions of Merlin with its binding partners other than DCAF1.     

Regulation of epithelial cell shape and polarity by cell - cell adhesion (Review)

Among all cell types that exhibit a polarized phenotype, epithelial cells are unique in that their polarity depends on the integration of the cell into a tissue, the epithelium. In recent years, the analysis of epithelial cell polarity in different epithelia and organisms has contributed to an understanding of the components involved and has further demonstrated that cell polarity and cell adhesion are intimately related to each other. Therefore, processes that mediate and modulate cell adhesion and coordinate adhesion and cell shape are fundamental for the function of epithelia. Recent results obtained in Drosophila melanogaster and Caenorhabditis elegans have provided further insight into the complex circuits regulating these processes, and have laid the direction for future analysis.