A New Membrane Protein Sbg1 Links the Contractile Ring Apparatus and Septum Synthesis Machinery in Fission Yeast.

Cytokinesis in many organisms requires a plasma membrane anchored actomyosin ring, whose contraction facilitates cell division. In yeast and fungi, actomyosin ring constriction is also coordinated with division septum assembly. How the actomyosin ring interacts with the plasma membrane and the plasma membrane-localized septum synthesizing machinery remains poorly understood. In Schizosaccharomyces pombe, an attractive model organism to study cytokinesis, the ß-1,3-glucan synthase Cps1p / Bgs1p, an integral membrane protein, localizes to the plasma membrane overlying the actomyosin ring and is required for primary septum synthesis. Through a high-dosage suppressor screen we identified an essential gene, sbg1+ (suppressor of beta glucan synthase 1), which suppressed the colony formation defect of Bgs1-defective cps1-191 mutant at higher temperatures. Sbg1p, an integral membrane protein, localizes to the cell ends and to the division site. Sbg1p and Bgs1p physically interact and are dependent on each other to localize to the division site. Loss of Sbg1p results in an unstable actomyosin ring that unravels and slides, leading to an inability to deposit a single contiguous division septum and an important reduction of the ß-1,3-glucan proportion in the cell wall, coincident with that observed in the cps1-191 mutant. Sbg1p shows genetic and / or physical interaction with Rga7p, Imp2p, Cdc15p, and Pxl1p, proteins known to be required for actomyosin ring integrity and efficient septum synthesis. This study establishes Sbg1p as a key member of a group of proteins that link the plasma membrane, the actomyosin ring, and the division septum assembly machinery in fission yeast.

Valine-glutamine (VQ) motif coding genes are ancient and non-plant-specific with comprehensive expression regulation by various biotic and abiotic stresses.

BACKGROUND: Valine-glutamine (VQ) motif containing proteins play important roles in abiotic and biotic stress responses in plants. However, little is known about the origin and evolution as well as comprehensive expression regulation of the VQ gene family. RESULTS: In this study, we systematically surveyed this gene family in 50 plant genomes from algae, moss, gymnosperm and angiosperm and explored their presence in other species from animals, bacteria, fungi and viruses. No VQs were detected in all tested algae genomes and all genomes from moss, gymnosperm and angiosperm encode varying numbers of VQs. Interestingly, some of fungi, lower animals and bacteria also encode single to a few VQs. Thus, they are not plant-specific and should be regarded as an ancient family. Their family expansion was mainly due to segmental duplication followed by tandem duplication and mobile elements. Limited contribution of gene conversion was detected to the family evolution. Generally, VQs were very much conserved in their motif coding region and were under purifying selection. However, positive selection was also observed during species divergence. Many VQs were up- or down-regulated by various abiotic / biotic stresses and phytohormones in rice and Arabidopsis. They were also co-expressed with some of other stress-related genes. All of the expression data suggest a comprehensive expression regulation of the VQ gene family. CONCLUSIONS: We provide new insights into gene expansion, divergence, evolution and their expression regulation of this VQ family. VQs were detectable not only in plants but also in some of fungi, lower animals and bacteria, suggesting the evolutionary conservation and the ancient origin. Overall, VQs are non-plant-specific and play roles in abiotic / biotic responses or other biological processes through comprehensive expression regulation.

Cylindrical cellular geometry ensures fidelity of division site placement in fission yeast.

Successful cytokinesis requires proper assembly of the contractile actomyosin ring, its stable positioning on the cell surface and proper constriction. Over the years, many of the key molecular components and regulators of the assembly and positioning of the actomyosin ring have been elucidated. Here we show that cell geometry and mechanics play a crucial role in the stable positioning and uniform constriction of the contractile ring. Contractile rings that assemble in locally spherical regions of cells are unstable and slip towards the poles. By contrast, actomyosin rings that assemble on locally cylindrical portions of the cell under the same conditions do not slip, but uniformly constrict the cell surface. The stability of the rings and the dynamics of ring slippage can be described by a simple mechanical model. Using fluorescence imaging, we verify some of the quantitative predictions of the model. Our study reveals an intimate interplay between geometry and actomyosin dynamics, which are likely to apply in a variety of cellular contexts.

The 14-3-3 protein rad24p modulates function of the cdc14p family phosphatase clp1p/flp1p in fission yeast.

Schizosaccharomyces pombe cells divide through the use of an actomyosin-based contractile ring. In response to perturbation of the actomyosin ring, S. pombe cells delay in a "cytokinesis-competent" state characterized by continuous repair and maintenance of the actomyosin ring and a G2 delay. This checkpoint mechanism requires the function of the Cdc14p-family phosphatase Clp1p/Flp1p and the septation initiation network (SIN). In response to cytokinetic defects, Clp1p, normally nucleolar in interphase, is retained in the cytoplasm until completion of cell division in a SIN-dependent manner. Here, we show that a phosphorylated form of Clp1p binds the 14-3-3 protein Rad24p and is retained in the cytoplasm in a Rad24p-dependent manner in response to cytokinesis defects. This physical interaction depends on the function of the SIN component, Sid2p. In the absence of Rad24p, cells are unable to maintain SIN signaling and lose viability upon mild cytokinetic stress. The requirement of Rad24p in this checkpoint is bypassed by ectopic activation of the SIN. Furthermore, SIN-dependent nuclear exclusion of Clp1p is dependent on Rad24p function. We conclude that Rad24p-mediated cytoplasmic retention of Clp1p/Flp1p is important for cell viability upon stress to the division apparatus.

Motor Activity Dependent and Independent Functions of Myosin II Contribute to Actomyosin Ring Assembly and Contraction in Schizosaccharomyces pombe.

Cytokinesis depends on a contractile actomyosin ring in many eukaryotes [1-3]. Myosin II is a key component of the actomyosin ring, although whether it functions as a motor or as an actin cross-linker to exert its essential role is disputed [1, 4, 5]. In Schizosaccharomyces pombe, the myo2-E1 mutation affects the upper 50 kDa sub-domain of the myosin II heavy chain, and cells carrying this lethal mutation are defective in actomyosin ring assembly at the non-permissive temperature [6, 7]. myo2-E1 also affects actomyosin ring contraction when rings isolated from permissive temperature-grown cells are incubated with ATP [8]. Here we report isolation of a compensatory suppressor mutation in the lower 50 kDa sub-domain (myo2-E1-Sup1) that reverses the inability of myo2-E1 to form colonies at the restrictive temperature. myo2-E1-Sup1 is capable of assembling normal actomyosin rings, although rings isolated from myo2-E1-Sup1 are defective in ATP-dependent contraction in vitro. Furthermore, the product of myo2-E1-Sup1 does not translocate actin filaments in motility assays in vitro. Superimposition of myo2-E1 and myo2-E1-Sup1 on available rigor and blebbistatin-bound myosin II structures suggests that myo2-E1-Sup1 may represent a novel actin translocation-defective allele. Actomyosin ring contraction and viability of myo2-E1-Sup1 cells depend on the late cytokinetic S. pombe myosin II isoform, Myp2p, a non-essential protein that is normally dispensable for actomyosin ring assembly and contraction. Our work reveals that Myo2p may function in two different and essential modes during cytokinesis: a motor activity-independent form that can promote actomyosin ring assembly and a motor activity-dependent form that supports ring contraction.

Insight into actin organization and function in cytokinesis from analysis of fission yeast mutants.

Actin is a key cytoskeletal protein with multiple roles in cellular processes such as polarized growth, cytokinesis, endocytosis, and cell migration. Actin is present in all eukaryotes as highly dynamic filamentous structures, such as linear cables and branched filaments. Detailed investigation of the molecular role of actin in various processes has been hampered due to the multifunctionality of the protein and the lack of alleles defective in specific processes. The actin cytoskeleton of the fission yeast, Schizosaccharomyces pombe, has been extensively characterized and contains structures analogous to those in other cell types. In this study, primarily with the view to uncover actin function in cytokinesis, we generated a large bank of fission yeast actin mutants that affect the organization of distinct actin structures and/or discrete physiological functions of actin. Our screen identified 17 mutants with specific defects in cytokinesis. Some of these cytokinesis mutants helped in dissecting the function of specific actin structures during ring assembly. Further genetic analysis of some of these actin mutants revealed multiple genetic interactions with mutants previously known to affect the actomyosin ring assembly. We also characterize a mutant allele of actin that is suppressed upon overexpression of Cdc8p-tropomyosin, underscoring the utility of this mutant bank. Another 22 mutant alleles, defective in polarized growth and/or other functions of actin obtained from this screen, are also described in this article. This mutant bank should be a valuable resource to study the physiological and biochemical functions of actin.

IQGAP-related Rng2p organizes cortical nodes and ensures position of cell division in fission yeast.

Correct positioning of the cell division machinery is crucial for genomic stability and cell fate determination. The fission yeast Schizosaccharomyces pombe, like animal cells, divides using an actomyosin ring and is an attractive model to study eukaryotic cytokinesis. In S. pombe, positioning of the actomyosin ring depends on the anillin-related protein Mid1p. Mid1p arrives first at the medial cortex and recruits actomyosin ring components to node-like structures, although how this is achieved is unknown. Here we show that the IQGAP-related protein Rng2p, an essential component of the actomyosin ring, is a key element downstream of Mid1p. Rng2p physically interacts with Mid1p and is required for the organization of other actomyosin ring components into cortical nodes. Failure of localization of Rng2p to the nodes prevents medial retention of Mid1p and leads to actomyosin ring assembly in a node-independent manner at nonmedial locations. We conclude that Mid1p recruits Rng2p to cortical nodes at the division site and that Rng2p, in turn, recruits other components of the actomyosin ring to cortical nodes, thereby ensuring correct placement of the division site.

SIN-inhibitory phosphatase complex promotes Cdc11p dephosphorylation and propagates SIN asymmetry in fission yeast.

BACKGROUND: Cytokinesis in many eukaryotes involves the function of an actomyosin-based contractile ring. In fission yeast, actomyosin ring maturation and stability require a conserved signaling pathway termed the SIN (septation initiation network). The SIN consists of a GTPase (Spg1p) and three protein kinases, all of which localize to the mitotic spindle pole bodies (SPBs). Two of the SIN kinases, Cdc7p and Sid1p, localize asymmetrically to the newly duplicated SPB in late anaphase. How this asymmetry is achieved is not understood, although it is known that their symmetric localization impairs cytokinesis. RESULTS: Here we characterize a new Forkhead-domain-associated protein, Csc1p, and identify SIN-inhibitory PP2A complex (SIP), which is crucial for the establishment of SIN asymmetry. Csc1p localizes to both SPBs early in mitosis, is lost from the SPB that accumulates Cdc7p, and instead accumulates at the SPB lacking Cdc7p. Csc1p is required for the dephosphorylation of the SIN scaffolding protein Cdc11p and is thereby required for the recruitment of Byr4p, a component of the GTPase-activating subunit for Spg1p, to the SPB. CONCLUSIONS: Because Cdc7p does not bind to GDP-Spg1p, we propose that the SIP-mediated Cdc11p dephosphorylation and the resulting recruitment of Byr4p are among the earliest steps in the establishment of SIN asymmetry.