The cell-end factor pom1p inhibits mid1p in specification of the cell division plane in fission yeast

Intrinsic spatial cues ensure the proper placement of the cell division plane. In the fission yeast Schizosaccharomyces pombe, the position of the nucleus helps to direct the medial positioning of contractile-ring assembly and subsequent cell division . An important factor in this process is mid1p (anillin-like protein), which is a peripheral-membrane protein that forms a broad cortical band of dots overlying the nucleus in interphase and recruits myosin in early mitosis . How mid1p localizes to this cortical band and tracks the nucleus is not clear, especially because its localization is independent of the cytoskeleton . Here, we used a combination of experimental and computational approaches to test mid1p localization mechanisms. We provide evidence that pom1p, a DYRK-family protein kinase that forms a concentration gradient emanating from the nongrowing cell end, inhibits mid1p. In pom1 mutants, mid1p is distributed over half of the cell, covering the nongrowing cell end. This abnormal distribution is established in a dynamic manner in interphase and leads to the formation of misplaced or multiple contractile rings. Our computational and experimental results support a model in which both positive cues from the medial nucleus and negative cues from the cell tips specify the position of the division plane.     

The integral membrane protein Pom34p functionally links nucleoporin subcomplexes

Here we have examined the function of Pom34p, a novel membrane protein in Saccharomyces cerevisiae, localized to nuclear pore complexes (NPCs). Membrane topology analysis revealed that Pom34p is a double-pass transmembrane protein with both the amino (N) and carboxy (C) termini positioned on the cytosolic/pore face. The network of genetic interactions between POM34 and genes encoding other nucleoporins was established and showed specific links between Pom34p function and Nup170p, Nup188p, Nup59p, Gle2p, Nup159p, and Nup82p. The transmembrane domains of Pom34p in addition to either the N- or C-terminal region were necessary for its function in different double mutants. We further characterized the pom34deltaN nup188delta mutant and found it to be perturbed in both NPC structure and function. Mislocalization of a subset of nucleoporins harboring phenylalanine-glycine repeats was observed, and nuclear import capacity for the Kap104p and Kap121p pathways was inhibited. In contrast, the pom34delta pom152delta double mutant was viable at all temperatures and showed no such defects. Interestingly, POM152 overexpression suppressed the synthetic lethality of pom34delta nup170delta and pom34delta nup59delta mutants. We speculate that multiple integral membrane proteins, either within the nuclear pore domain or in the nuclear envelope, execute coordinated roles in NPC structure and function.     

Cell division: mid-level management

When a fission yeast cell divides, the anillin-like protein mid1p helps to position the contractile ring in the cell middle. Recent experiments from two groups have shown how the cell-polarity factor pom1p negatively regulates the distribution of mid1p.     

A tip-high gradient of a putative plasma membrane SNARE approximates the exocytotic gradient in hyphal apices of the fungus Neurospora crassa

Antibodies to the Saccharomyces cereviseae plasma membrane t-SNARE Sso2p identify a putative 39-kDa homologue in Neurospora crassa. The 39-kDa protein is enriched in plasma membrane (PM) and occurred with other membranes. It is extractable by detergent, but not chaotropic or alkali agents, suggesting membrane insertion. Immunoprecipitation with anti-Sso2p coprecipitated a approximately 100-kDa, Mg(+)-ATP-sensitive band with the 39-kDa protein, suggesting a ternary SNARE complex. Affinity-purified anti-Sso2p gave hyphal staining patterns most consistent with protein localization on both the PM and intracellular exocytotic apical wall vesicles. The PM staining in hyphal apices formed a tip-high gradient, not as steep as that predicted by the "hyphoid equation," but closer to published gradients of cell wall matrix deposition. We conclude that the t-SNAREs are transported to the PM on the apical vesicles, but their tip-high gradient alone is insufficient to explain the vesicle fusion gradient in growing tips.     

A phosphorylation cycle shapes gradients of the DYRK family kinase Pom1 at the plasma membrane

Concentration gradients regulate many cell biological and developmental processes. In rod-shaped fission yeast cells, polar cortical gradients of the DYRK family kinase Pom1 couple cell length with mitotic commitment by inhibiting a mitotic inducer positioned at midcell. However, how Pom1 gradients are established is unknown. Here, we show that Tea4, which is normally deposited at cell tips by microtubules, is both necessary and, upon ectopic cortical localization, sufficient to recruit Pom1 to the cell cortex. Pom1 then moves laterally at the plasma membrane, which it binds through a basic region exhibiting direct lipid interaction. Pom1 autophosphorylates in this region to lower lipid affinity and promote membrane release. Tea4 triggers Pom1 plasma membrane association by promoting its dephosphorylation through the protein phosphatase 1 Dis2. We propose that local dephosphorylation induces Pom1 membrane association and nucleates a gradient shaped by the opposing actions of lateral diffusion and autophosphorylation-dependent membrane detachment.     

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.     

Cell-to-cell movement of Potato virus X: the role of p12 and p8 encoded by the second and third open reading frames of the triple gene block

Potato virus X (PVX) requires three proteins, p25, p12, and p8, encoded by the triple gene block plus the coat protein (CP) for cell-to-cell movement. When each of these proteins was co-expressed with a cytosolic green fluorescent protein (GFP) in the epidermal cells of Nicotiana benthamiana by the microprojectile bombardment-mediated gene delivery method, only p12 enhanced diffusion of co-expressed GFP, indicating an ability to alter plasmodesmal permeability. p25, p12, and CP, expressed transiently in the initially infected cells, transcomplemented the corresponding movement-defective mutants to spread through two or more cell boundaries. Thus, these proteins probably move from cell to cell with the genomic RNA. In contrast, p8 only functioned intracellularly and was not absolutely required for cell-to-cell movement. Since overexpression of p12 overcame the p8 deficiency, p8 appears to facilitate the functioning of p12, presumably by mediating its intracellular trafficking. Considering the likelihood that p12 and p8 are membrane proteins, it is suggested that intercellular as well as intracellular movement of PVX involves a membrane-mediated process.     

An important role for syndecan-1 in herpes simplex virus type-1 induced cell-to-cell fusion and virus spread

Herpes simplex virus type-1 (HSV-1) is a common human pathogen that relies heavily on cell-to-cell spread for establishing a lifelong latent infection. Molecular aspects of HSV-1 entry into host cells have been well studied; however, the molecular details of the spread of the virus from cell-to-cell remain poorly understood. In the past, the role of heparan sulfate proteoglycans (HSPG) during HSV-1 infection has focused solely on the role of HS chains as an attachment receptor for the virus, while the core protein has been assumed to perform a passive role of only carrying the HS chains. Likewise, very little is known about the involvement of any specific HSPGs in HSV-1 lifecycle. Here we demonstrate that a HSPG, syndecan-1, plays an important role in HSV-1 induced membrane fusion and cell-to-cell spread. Interestingly, the functions of syndecan-1 in fusion and spread are independent of the presence of HS on the core protein. Using a mutant CHO-K1 cell line that lacks all glycosaminoglycans (GAGs) on its surface (CHO-745) we demonstrate that the core protein of syndecan-1 possesses the ability to modulate membrane fusion and viral spread. Altogether, we identify a new role for syndecan-1 in HSV-1 pathogenesis and demonstrate HS-independent functions of its core protein in viral spread.     

Isolation and assay of corn root membrane vesicles with reduced proton permeability

Conditions promoting the formation of sealed membrane vesicles from corn roots with reduced proton permeability were examined using the probe 9-aminoacridine as a rapid indicator of pH gradient formation and dissipation. Plasma membrane vesicles isolated by differential and density gradient centrifugation were leaky to protons and rapidly equilibrated when exposed to artificially imposed pH gradients. The leaky plasma membrane vesicles showed reduced proton permeability when incubated with calcium or with excess phospholipids. However, these vesicles were unable to form ATP-induced pH gradients. Sealed vesicles isolated by discontinuous Ficoll gradient centrifugation of a microsomal fraction displayed reduced proton permeability and were osmotically active. In contrast to purified plasma membrane vesicles, the microsomal-derived vesicles were more suitable for studies of active proton transport.     

The integral membrane nucleoporin pom121 functionally links nuclear pore complex assembly and nuclear envelope formation

The metazoan nuclear envelope (NE) breaks down and reforms at each mitosis. Nuclear pore complexes (NPCs), which allow nucleocytoplasmic transport during interphase, assemble into the reforming NE at the end of mitosis. Using in vitro NE assembly assays, we show that one of the two transmembrane nucleoporins, pom121, is essential for NE formation, whereas the second, gp210, is dispensable. Depletion of either pom121-containing membrane vesicles or the protein alone does not affect vesicle binding to chromatin but prevents their fusion to form a closed NE. When the Nup107-160 complex, which is essential for integration of NPCs into the NE, is also depleted, pom121 becomes dispensable for NE formation, suggesting a close functional link between NPC and NE formation and the existence of a checkpoint that monitors NPC assembly state.     

Precision of sensing cell length via concentration gradients

Unicellular organisms are typically found to have a characteristic cell size. To achieve a homeostatic distribution of cell sizes over many generations requires that cell length is actively sensed and regulated. However, the mechanisms by which cell size is controlled remain poorly understood. Recent experiments in fission yeast have shown that cell length is controlled in part by polar gradients of the protein Pom1 together with localized measurement of concentration at midcell. Dilution as the cell grows leads to a reduction in the midcell protein concentration, which lifts a block on mitosis. Here we analyze the precision of this mechanism for length sensing in the presence of inevitable intrinsic noise in the processes leading to formation and measurement of this gradient. We find that the use of concentration gradients allows for more robust length sensing than a comparable spatially uniform system, and allows for reliable length determination even if the average protein concentration throughout the cell remains constant as the cell grows. Optimal values for the gradient decay length and receptor dissociation constant emerge from maximizing sensitivity while minimizing the impact of density fluctuations.     

Compensation effect of the MAPK cascade on formation of phospho-protein gradients

The signal-transfer process in the mitogen-activated protein kinase (MAPK) cascade is formulated as a reaction-diffusion system describing the complete three-step phospho-protein reactions and the diffusion process in the direction from the cell membrane to the nucleus. The simulation analysis of the model demonstrates that MAPK cascade can work as a signal amplifier so as to compensate the signal attenuation due to formation of phospho-protein gradients. It also is found to be attainable for eukaryotic cells that a steep gradient of phosphorylated MAPK is not formed in a certain range of the system parameter values. One of the distinctive features in the formation of phospho-protein gradients is revealed to be its high sensitivity to a change in parameter values such as diffusion distance, diffusion coefficients and enzymatic activities of the phosphatases, suggesting that these parameters may act as the key factors for regulation of the signal transduction systems.     

Spatial gradients of cellular phospho-proteins.

If a protein is rapidly phosphorylated and dephosphorylated at separate cellular locations and protein diffusion is slow, then a spatial gradient of the phosphorylated form of the protein may develop within the cell. We have estimated the potential size of such gradients using measured values of protein diffusion coefficients and protein kinase and phosphatase activities. We analysed two different cellular geometries: (1) where the kinases is located on the plasma membrane of a spherical cell and the phospatase is distributed homogenously in the cytoplasm and (2) where the kinase is located on one planar membrane and the phosphatase on a second parallel planar membrane. The estimated gradients of phospho-proteins were potentially very large, which has important implications for cellular signalling.     

Formation of steady-state oxygen gradients in vitro: application to liver zonation

We have developed a perfusion bioreactor system that allows the formation of steady state oxygen gradients in cell culture. In this study, gradients were formed in cultures of rat hepatocytes to study the role of oxygen in modulating cellular functions. A model of oxygen transport in our flat-plate reactor was developed to estimate oxygen distribution at the cell surface. Experimental measurements of outlet oxygen concentration from various flow conditions were used to validate model predictions. We showed that cell viability was maintained over a 24-h period when operating with a physiologic oxygen gradient at the cell surface from 76 to 5 mmHg O(2) at the outlet. Oxygen gradients have been implicated in the maintenance of regional compartmentalized metabolic and detoxification functions in the liver, termed zonation. In this system, physiologic oxygen gradients in reactor cultures contributed to a heterogeneous distribution of phosphoenolpyruvate carboxykinase (predominantly localized upstream) and cytochrome p450 2B (predominantly localized downstream) that correlates with the distribution of these enzymes in vivo. The oxygen gradient chamber provides a means of probing the oxygen effects in vitro over a continuous range of O(2) tensions. In addition, this system serves as an in vitro model of zonation that could be further extended to study the role of gradients in ischemia-reperfusion injury, toxicity, and bioartificial liver design.     

The scaffolding protein Dlg1 is a negative regulator of cell-free virus infectivity but not of cell-to-cell HIV-1 transmission in T cells

Background

Cell-to-cell virus transmission of Human immunodeficiency virus type-1 (HIV-1) is predominantly mediated by cellular structures such as the virological synapse (VS). The VS formed between an HIV-1-infected T cell and a target T cell shares features with the immunological synapse (IS). We have previously identified the human homologue of the Drosophila Discs Large (Dlg1) protein as a new cellular partner for the HIV-1 Gag protein and a negative regulator of HIV-1 infectivity. Dlg1, a scaffolding protein plays a key role in clustering protein complexes in the plasma membrane at cellular contacts. It is implicated in IS formation and T cell signaling, but its role in HIV-1 cell-to-cell transmission was not studied before.

Methodology/Principal Findings

Kinetics of HIV-1 infection in Dlg1-depleted Jurkat T cells show that Dlg1 modulates the replication of HIV-1. Single-cycle infectivity tests show that this modulation does not take place during early steps of the HIV-1 life cycle. Immunofluorescence studies of Dlg1-depleted Jurkat T cells show that while Dlg1 depletion affects IS formation, it does not affect HIV-1-induced VS formation. Co-culture assays and quantitative cell-to-cell HIV-1 transfer analyses show that Dlg1 depletion does not modify transfer of HIV-1 material from infected to target T cells, or HIV-1 transmission leading to productive infection via cell contact. Dlg1 depletion results in increased virus yield and infectivity of the viral particles produced. Particles with increased infectivity present an increase in their cholesterol content and during the first hours of T cell infection these particles induce higher accumulation of total HIV-1 DNA.

Conclusion

Despite its role in the IS formation, Dlg1 does not affect the VS and cell-to-cell spread of HIV-1, but plays a role in HIV-1 cell-free virus transmission. We propose that the effect of Dlg1 on HIV-1 infectivity is at the stage of virus entry.     

Conjugal transfer replication of R64drd11 plasmid DNA in the donor cells of Escherichia coli K-12

Summary Conjugation, the process of genetic transfer requiring cell-to-cell contact, has been the focus of many investigations. In recent years, the molecular aspect of conjugation has been questioned. Since it has been shown that during exponential growth plasmid DNA forms a complex with the folded chromosomal complex (FCC), the relationship of R64drd11 plasmid DNA to the FCC (chromosome plus membrane) during conjugal replication was examined. A cell system was used which allowed specific observation of conjugal events as they occurred in the donor cell. Evidence is presented to show that conjugally replicating R64drd11 covalently closed circular molecules co-sediment with the FCC in neutral sucrose gradients. The use of density gradients to separate DNA from membrane-bound DNA from free membrane, indicate that the membrane is the preferential structure for conjugally replicating plasmid DNA association.     

Nuclear pore complex function in Saccharomyces cerevisiae is influenced by glycosylation of the transmembrane nucleoporin Pom152p

The regulated transport of proteins across the nuclear envelope occurs through nuclear pore complexes (NPCs), which are composed of >30 different protein subunits termed nucleoporins. While some nucleoporins are glycosylated, little about the role of glycosylation in NPC activity is understood. We have identified loss-of-function alleles of ALG12, encoding a mannosyltransferase, as suppressors of a temperature-sensitive mutation in the gene encoding the FXFG-nucleoporin NUP1. We observe that nup1Delta cells import nucleophilic proteins more efficiently when ALG12 is absent, suggesting that glycosylation may influence nuclear transport. Conditional nup1 and nup82 mutations are partially suppressed by the glycosylation inhibitor tunicamycin, while nic96 and nup116 alleles are hypersensitive to tunicamycin treatment, further implicating glycosylation in NPC function. Because Pom152p is a glycosylated, transmembrane nucleoporin, we examined genetic interactions between pom152 mutants and nup1Delta. A nup1 deletion is lethal in combination with pom152Delta, as well as with truncations of the N-terminal and transmembrane regions of Pom152p. However, truncations of the N-glycosylated, lumenal domain of Pom152p and pom152 mutants lacking N-linked glycosylation sites are viable in combination with nup1Delta, suppress nup1Delta temperature sensitivity, and partially suppress the nuclear protein import defects associated with the deletion of NUP1. These data provide compelling evidence for a role for glycosylation in influencing NPC function.     

Functional Analysis of Vaccinia Virus B5R Protein: Essential Role in Virus Envelopment Is Independent of a Large Portion of the Extracellular Domain

Vaccinia virus has two forms of infectious virions: the intracellular mature virus and the extracellular enveloped virus (EEV). EEV is critical for cell-to-cell and long-range spread of the virus. The B5R open reading frame (ORF) encodes a membrane protein that is essential for EEV formation. Deletion of the B5R ORF results in a dramatic reduction of EEV, and as a consequence, the virus produces small plaques in vitro and is highly attenuated in vivo. The extracellular portion of B5R is composed mainly of four domains that are similar to the short consensus repeats (SCRs) present in complement regulatory proteins. To determine the contribution of these putative SCR domains to EEV formation, we constructed recombinant vaccinia viruses that replaced the wild-type B5R gene with a mutated gene encoding a B5R protein lacking the SCRs. The resulting recombinant viruses produced large plaques, indicating efficient cell-to-cell spread in vitro, and gradient centrifugation of supernatants from infected cells confirmed that EEV was formed. In contrast, phalloidin staining of infected cells showed that the virus lacking the SCR domains was deficient in the induction of thick actin bundles. Thus, the highly conserved SCR domains present in the extracellular portion of the B5R protein are dispensable for EEV formation. This indicates that the B5R protein is a key viral protein with multiple functions in the process of virus envelopment and release. In addition, given the similarity of the extracellular domain to complement control proteins, the B5R protein may be involved in viral evasion from host immune responses.     

Association of sequestered beta-adrenergic receptors with the plasma membrane: a novel mechanism for receptor down regulation

Chronic exposure of frog erythrocytes to beta-adrenergic agonists leads to desensitization of the responsiveness of adenylate cyclase to isoproterenol and is accompanied by "down-regulation", a decrease in the number of beta-adrenergic receptors on the cell surface. When frog erythrocyte plasma membranes are prepared by osmotic lysis of cells, the receptors lost from the cell surface during desensitization can be recovered in a "light membrane fraction", obtained by centrifuging the cell cytosol at 158,000 X g for 1 hr. These receptors are sequestered away from the plasma membrane fraction which contains the adenylate cyclase and the guanine nucleotide regulatory protein. If desensitized frog erythrocytes are disrupted by gentler freeze/thaw procedures, however, the sequestered beta-adrenergic receptors can be demonstrated to be physically associated with the plasma membrane. Typically, plasma membranes prepared in this fashion do not demonstrate a significant down regulation despite attenuation of isoproterenol-stimulated adenylate cyclase activity. Under these conditions, beta-adrenergic receptors from control and desensitized preparations co-migrate on sucrose density gradients in exactly the same place as the plasma membrane marker, adenylate cyclase. In contrast, when membranes from osmotically lysed desensitized cells are fractionated on sucrose gradients the down regulated receptors are sequestered in a light membrane fraction which barely enters the gradient and which is physically separated from adenylate cyclase activity. The data are consistent with a novel mechanism of receptor down-regulation which appears to involve the sequestration of the beta-adrenergic receptors away from the cell surface into a membrane compartment which remains physically associated with the plasma membrane.     

Pom1 gradient buffering through intermolecular auto‐phosphorylation

Concentration gradients provide spatial information for tissue patterning and cell organization, and their robustness under natural fluctuations is an evolutionary advantage. In rod‐shaped Schizosaccharomyces pombe cells, the DYRK‐family kinase Pom1 gradients control cell division timing and placement. Upon dephosphorylation by a Tea4‐phosphatase complex, Pom1 associates with the plasma membrane at cell poles, where it diffuses and detaches upon auto‐phosphorylation. Here, we demonstrate that Pom1 auto‐phosphorylates intermolecularly, both in vitro and in vivo, which confers robustness to the gradient. Quantitative imaging reveals this robustness through two system's properties: The Pom1 gradient amplitude is inversely correlated with its decay length and is buffered against fluctuations in Tea4 levels. A theoretical model of Pom1 gradient formation through intermolecular auto‐phosphorylation predicts both properties qualitatively and quantitatively. This provides a telling example where gradient robustness through super‐linear decay, a principle hypothesized a decade ago, is achieved through autocatalysis. Concentration‐dependent autocatalysis may be a widely used simple feedback to buffer biological activities.