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.     

Distinct levels in Pom1 gradients limit Cdr2 activity and localization to time and position division

Where and when cells divide are fundamental questions. In rod-shaped fission yeast cells, the DYRK-family kinase Pom1 is organized in concentration gradients from cell poles and controls cell division timing and positioning. Pom1 gradients restrict to mid-cell the SAD-like kinase Cdr2, which recruits Mid1/Anillin for medial division. Pom1 also delays mitotic commitment through Cdr2, which inhibits Wee1. Here, we describe quantitatively the distributions of cortical Pom1 and Cdr2. These reveal low profile overlap contrasting with previous whole-cell measurements and Cdr2 levels increase with cell elongation, raising the possibility that Pom1 regulates mitotic commitment by controlling Cdr2 medial levels. However, we show that distinct thresholds of Pom1 activity define the timing and positioning of division. Three conditions—a separation-of-function Pom1 allele, partial downregulation of Pom1 activity, and haploinsufficiency in diploid cells—yield cells that divide early, similar to pom1 deletion, but medially, like wild-type cells. In these cells, Cdr2 is localized correctly at mid-cell. Further, Cdr2 overexpression promotes precocious mitosis only in absence of Pom1. Thus, Pom1 inhibits Cdr2 for mitotic commitment independently of regulating its localization or cortical levels. Indeed, we show Pom1 restricts Cdr2 activity through phosphorylation of a C-terminal self-inhibitory tail. In summary, our results demonstrate that distinct levels in Pom1 gradients delineate a medial Cdr2 domain, for cell division placement, and control its activity, for mitotic commitment.     

Direct and indirect regulation of Pom1 cell size pathway by the protein phosphatase 2C Ptc1

The fission yeast cells Schizosaccharomyces pombe divide at constant cell size regulated by environmental stimuli. An important pathway of cell size control involves the membrane-associated DYRK-family kinase Pom1, which forms decreasing concentration gradients from cell poles and inhibits mitotic inducers at midcell. Here, we identify the phosphatase 2C Ptc1 as negative regulator of Pom1. Ptc1 localizes to cell poles in a manner dependent on polarity and cell-wall integrity factors. We show that Ptc1 directly binds Pom1 and can dephosphorylate it in vitro but modulates Pom1 localization indirectly upon growth in low-glucose conditions by influencing microtubule stability. Thus, Ptc1 phosphatase plays both direct and indirect roles in the Pom1 cell size control pathway.     

Noise reduction in the intracellular pom1p gradient by a dynamic clustering mechanism

Chemical gradients can generate pattern formation in biological systems. In the fission yeast Schizosaccharomyces pombe, a cortical gradient of pom1p (a DYRK-type protein kinase) functions to position sites of cytokinesis and cell polarity and to control cell length. Here, using quantitative imaging, fluorescence correlation spectroscopy, and mathematical modeling, we study how its gradient distribution is formed. Pom1p gradients exhibit large cell-to-cell variability, as well as dynamic fluctuations in each individual gradient. Our data lead to a two-state model for gradient formation in which pom1p molecules associate with the plasma membrane at cell tips and then diffuse on the membrane while aggregating into and fragmenting from clusters, before disassociating from the membrane. In contrast to a classical one-component gradient, this two-state gradient buffers against cell-to-cell variations in protein concentration. This buffering mechanism, together with time averaging to reduce intrinsic noise, allows the pom1p gradient to specify positional information in a robust manner.     

Pom1 DYRK regulates localization of the Rga4 GAP to ensure bipolar activation of Cdc42 in fission yeast

BACKGROUND: In the fission yeast Schizosaccharomyces pombe, cell growth takes place exclusively at both ends of the cylindrical cell. During this highly polarized growth, microtubules are responsible for the placement of the cell-end marker proteins, the Tea1-Tea4/Wsh3 complex, which recruits the Pom1 DYRK-family protein kinase. Pom1 is required for proper positioning of growth sites, and the Deltapom1 mutation brings about monopolar cell growth. RESULTS: Pom1 kinase physically interacts with Rga4, which has a GAP (GTPase-activating protein) domain for Rho-family GTPase. Genetic and biochemical evidence indicates that Rga4 functions as GAP for the Cdc42 GTPase, an evolutionarily conserved regulator of F-actin. CRIB (Cdc42/Rac interactive binding)-GFP microscopy has revealed that GTP-bound, active Cdc42 is concentrated to growing cell ends accompanied by developed F-actin structures, where the Rga4 GAP is excluded. The monopolar Deltapom1 mutant fails to eliminate Rga4 from the nongrowing cell end, resulting in monopolar distribution of GTP-Cdc42 to the growing cell end. However, mutational inactivation of Rga4 allows Cdc42 to be active at both ends of Deltapom1 cells, suggesting that mislocalization of Rga4 in the Deltapom1 mutant contributes to its monopolar phenotype. CONCLUSIONS: Pom1 kinase recruited to cell ends by the Tea1-Tea4/Wsh3 complex is essential for proper localization of a GAP for Cdc42, Rga4, which ensures bipolar localization of GTP-bound, active Cdc42. Because of the established role of Cdc42 in F-actin formation, these observations provide a new insight into how the microtubule system achieves localized formation of F-actin to generate cell polarity.     

A Highlights from MBoC Selection: Megadalton-node assembly by binding of Skb1 to the membrane anchor Slf1

The plasma membrane contains both dynamic and static microdomains. Given the growing appreciation of cortical microdomains in cell biology, it is important to determine the organizational principles that underlie assembly of compartmentalized structures at the plasma membrane. The fission yeast plasma membrane is highly compartmentalized by distinct sets of cortical nodes, which control signaling for cell cycle progression and cytokinesis. The mitotic inhibitor Skb1 localizes to a set of cortical nodes that provide spatial control over signaling for entry into mitosis. However, it has been unclear whether these nodes contain other proteins and how they might be organized and tethered to the plasma membrane. Here we show that Skb1 forms nodes by interacting with the novel protein Slf1, which is a limiting factor for node formation in cells. Using quantitative fluorescence microscopy and in vitro assays, we demonstrate that Skb1-Slf1 nodes are megadalton structures that are anchored to the membrane by a lipid-binding region in the Slf1 C-terminus. We propose a mechanism for higher-order node formation by Skb1 and Slf1, with implications for macromolecular assemblies in diverse cell types.     

Wsh3/Tea4 is a novel cell-end factor essential for bipolar distribution of Tea1 and protects cell polarity under environmental stress in S. pombe

BACKGROUND: The fission yeast Schizosaccharomyces pombe has a cylindrical cell shape, for which growth is strictly limited to both ends, and serves as an excellent model system for genetic analysis of cell-polarity determination. Previous studies identified a cell-end marker protein, Tea1, that is transported by cytoplasmic microtubules to cell tips and recruits other cell-end factors, including the Dyrk-family Pom1 kinase. The deltatea1 mutant cells cannot grow in a bipolar fashion and show T-shaped morphology after heat shock. RESULTS: We identified Wsh3/Tea4 as a novel protein that interacts with Win1 MAP kinase kinase kinase (MAPKKK) of the stress-activated MAP kinase cascade. Wsh3 forms a complex with Tea1 and is transported to cell tips by growing microtubules. The deltawsh3 mutant shows monopolar growth with abnormal Tea1 aggregate at the non-growing cell end; this abnormal aggregate fails to recruit Pom1 kinase. Consistent with the observed interaction between Win1 and Wsh3, cells lacking Wsh3 or Tea1 show more severe cell-polarity defects under osmolarity and heat-stress stimuli that are known to activate the stress MAPK cascade. Furthermore, mutants of the stress MAPK also exhibit cell-polarity defects when exposed to the same stress. CONCLUSIONS: Wsh3/Tea4 is an essential component of the Tea1 cell-end complex. In addition to its role in bipolar growth during the normal cell cycle, the Wsh3-Tea1 complex, together with the stress-signaling MAPK cascade, contributes to cell-polarity maintenance under stress conditions.     

Quantitative Phosphoproteomics Reveals Pathways for Coordination of Cell Growth and Division by the Conserved Fission Yeast Kinase Pom1

Complex phosphorylation-dependent signaling networks underlie the coordination of cellular growth and division. In the fission yeast Schizosaccharomyces pombe, the Dual specificity tyrosine-(Y)-phosphorylation regulated kinase (DYRK) family protein kinase Pom1 regulates cell cycle progression through the mitotic inducer Cdr2 and controls cell polarity through unknown targets. Here, we sought to determine the phosphorylation targets of Pom1 kinase activity by SILAC-based phosphoproteomics. We defined a set of high-confidence Pom1 targets that were enriched for cytoskeletal and cell growth functions. Cdr2 was the only cell cycle target of Pom1 kinase activity that we identified in cells. Mutation of Pom1-dependent phosphorylation sites in the C terminus of Cdr2 inhibited mitotic entry but did not impair Cdr2 localization. In addition, we found that Pom1 phosphorylated multiple substrates that function in polarized cell growth, including Tea4, Mod5, Pal1, the Rho GAP Rga7, and the Arf GEF Syt22. Purified Pom1 phosphorylated these cell polarity targets in vitro, confirming that they are direct substrates of Pom1 kinase activity and likely contribute to regulation of polarized growth by Pom1. Our study demonstrates that Pom1 acts in a linear pathway to control cell cycle progression while regulating a complex network of cell growth targets.The coordination of cell growth and division represents a fundamental concept in cell biology. The mechanisms that promote polarized growth and drive cell cycle progression are complex signaling networks that operate in a wide range of cell types and organisms. Understanding these networks and their molecular connections requires large-scale approaches that define the underlying biochemical reactions. Phosphorylation drives many events in both cell polarity and cell cycle signaling, and protein kinases that act in both processes represent key players in coordinated growth and division.The fission yeast S. pombe has served as a long-standing model organism for studies on cell polarity and the cell cycle. The fission yeast protein kinase Pom1 is an intriguing candidate to function in the coordination of polarized growth and cell cycle progression. This DYRK¹ family kinase was originally identified as a polarity mutant (hence the name Pom1) in a genetic screen for misshapen cells (1). Later studies revealed an additional role for Pom1 in cell cycle progression, where it delays mitotic entry until cells reach a critical size threshold (2, 3). Thus, pom1Δ mutant cells display defects in both cell polarity and cell size at mitosis, as well as misplaced division septa (1–6). Mutations that impair Pom1 kinase activity mimic these deletion phenotypes, indicating a key role for Pom1-dependent phosphorylation. The pleiotropic phenotype of pom1 mutants might result from Pom1 phosphorylating distinct substrates for cell polarity versus mitotic entry, but the targets of Pom1 kinase activity are largely unknown. Only two Pom1 substrates have been identified to date. First, Pom1 auto-phosphorylates as part of a mechanism that promotes localization in a cortical gradient enriched at cell tips (7). Second, Pom1 phosphorylates two regions of the protein kinase Cdr2. Phosphorylation of Cdr2 C terminus is proposed to prevent mitotic entry by inhibiting Cdr2 kinase activity (8, 9), while phosphorylation near membrane-binding motifs of Cdr2 promotes medial cell division by inhibiting localization of Cdr2 at cell tips (10). It has been unclear if Cdr2 represents the only cell cycle target of Pom1 kinase activity, and no cell polarity targets of Pom1 have been identified. In order to clarify how this protein kinase controls multiple cellular processes, we have comprehensively cataloged Pom1 substrates by quantitative phosphoproteomics. Such a large-scale approach also has the potential to reveal general mechanisms that operate in the coordination of cell growth and division.Stable isotope labeling of amino acids in culture (SILAC) combined with phosphopeptide enrichment and mass spectrometry has allowed the proteome-wide analysis of protein phosphorylation from diverse experimental systems (11–15). In this approach, cells are grown separately in media containing normal (“light”) or isotope-labeled (“heavy”) arginine and lysine, treated, mixed, and processed for LC-MS/MS analysis. In combination with analog-sensitive protein kinase mutants, which can be rapidly and specifically inhibited by nonhydrolyzable ATP analogs (16, 17), SILAC presents a powerful approach to identify cellular phosphorylation events that depend on a specific protein kinase. This method is particularly well suited for studies in yeast, where analog-sensitive protein kinase mutants can be readily integrated into the genome.In this study, we have employed SILAC-based phosphoproteomics to identify Pom1 substrates in fission yeast. New Pom1 targets were verified as direct substrates in vitro, and our analysis indicates that Pom1 controls cell cycle progression through a single target while coordinating a more complex network of cell polarity targets.     

Tea3p is a cell end marker activating polarized growth in Schizosaccharomyces pombe

Eukaryotic cells are often polarized in their cytoplasmic structures, and this can be important for their function. The fission yeast Schizosaccharomyces pombe is a highly polarized cell that extends bipolarly along a single axis to generate a rod-shaped cell. It divides by medial fission to generate two equal-sized daughter cells that resume growth only at the old end. Once these cells have reached a particular length, they undergo NETO, new end take-off, whereby growth is activated at the other end to generate bipolarly extending cells. The activation and positioning of these growth zones are essential for maintaining growth in a straight line. Genetic analyses have identified many proteins involved in this process, like the cell end markers Tea1p and Pom1p and the kinases Orb2p/Shk1p/Pak1, Ssp1p, and Wee1p. Here, we describe tea3, a gene encoding a tea1-like protein with some similarities to ERM proteins. Tea3p is required for efficient NETO and for the proper placement of the septum. Like Pom1p, Tea3p localizes to cell ends, and its localization depends on microtubules and Tea1p. We propose that Tea3p is a novel cell end marker required specifically to activate polarized cell growth at the second end during NETO.     

Dynamic regulation of mammalian numb by G protein-coupled receptors and protein kinase C activation: Structural determinants of numb association with the cortical membrane

The cell fate determinant Numb is a membrane-associated adaptor protein involved in both development and intracellular vesicular trafficking. It has a phosphotyrosine-binding (PTB) domain and COOH-terminal endocytic-binding motifs for alpha-adaptin and Eps15 homology domain-containing proteins. Four isoforms of Numb are expressed in vertebrates, two of which selectively associate with the cortical membrane. In this study, we have characterized a cortical pool of Numb that colocalizes with AP2 and Eps15 at substratum plasma membrane punctae and cortical membrane-associated vesicles. Green fluorescent protein (GFP)-tagged mutants of Numb were used to identify the structural determinants required for localization. In addition to the previously described association of the PTB domain with the plasma membrane, we show that the AP2-binding motifs facilitate the association of Numb with cortical membrane punctae and vesicles. We also show that agonist stimulation of G protein-coupled receptors (GPCRs) that are linked to phospholipase Cbeta and protein kinase C (PKC) activation causes redistribution of Numb from the cortical membrane to the cytosol. This effect is correlated with Numb phosphorylation and an increase in its Triton X-100 solubility. Live-imaging analysis of mutants identified two regions within Numb that are independently responsive to GPCR-mediated lipid hydrolysis and PKC activation: the PTB domain and a region encompassing at least three putative PKC phosphorylation sites. Our data indicate that membrane localization of Numb is dynamically regulated by GPCR-activated phospholipid hydrolysis and PKC-dependent phosphorylation events.     

Polarity determinants Tea1p, Tea4p, and Pom1p inhibit division-septum assembly at cell ends in fission yeast

Correct positioning of the cell-division plane is crucial for cell function in all organisms. The fission yeast Schizosaccharomyces pombe divides by utilizing an actomyosin-based contractile ring and is an attractive model for the study of cytokinesis. The metazoan anillin-related protein Mid1p stimulates medial assembly of the division septum by recruiting actomyosin-ring components to the medial cortex. Here, we describe an inhibitory mechanism, involving the cell-end-localized polarity determinants Tea1p, Tea4p/Wsh3p, and Pom1p (tip complex), which prevents division-septum assembly at the cell ends. While Mid1p and the tip complex are dispensable for cell viability, their simultaneous loss leads to lethality. The FER/CIP homology protein Cdc15p, which organizes the actomyosin ring and cell membranes during cytokinesis, is a candidate for regulation by the tip complex. Since dual regulation of division-site placement is also seen in nematodes, such regulation might be a general feature of eukaryotic cytokinesis.     

A Highlights from MBoC Selection: Compartmentalized nodes control mitotic entry signaling in fission yeast

Cell cycle progression is coupled to cell growth, but the mechanisms that generate growth-dependent cell cycle progression remain unclear. Fission yeast cells enter into mitosis at a defined size due to the conserved cell cycle kinases Cdr1 and Cdr2, which localize to a set of cortical nodes in the cell middle. Cdr2 is regulated by the cell polarity kinase Pom1, suggesting that interactions between cell polarity proteins and the Cdr1-Cdr2 module might underlie the coordination of cell growth and division. To identify the molecular connections between Cdr1/2 and cell polarity, we performed a comprehensive pairwise yeast two-hybrid screen. From the resulting interaction network, we found that the protein Skb1 interacted with both Cdr1 and the Cdr1 inhibitory target Wee1. Skb1 inhibited mitotic entry through negative regulation of Cdr1 and localized to both the cytoplasm and a novel set of cortical nodes. Skb1 nodes were distinct structures from Cdr1/2 nodes, and artificial targeting of Skb1 to Cdr1/2 nodes delayed entry into mitosis. We propose that the formation of distinct node structures in the cell cortex controls signaling pathways to link cell growth and division.     

Molecular networks linked by Moesin drive remodeling of the cell cortex during mitosis

The cortical mechanisms that drive the series of mitotic cell shape transformations remain elusive. In this paper, we identify two novel networks that collectively control the dynamic reorganization of the mitotic cortex. We demonstrate that Moesin, an actin/membrane linker, integrates these two networks to synergize the cortical forces that drive mitotic cell shape transformations. We find that the Pp1-87B phosphatase restricts high Moesin activity to early mitosis and down-regulates Moesin at the polar cortex, after anaphase onset. Overactivation of Moesin at the polar cortex impairs cell elongation and thus cytokinesis, whereas a transient recruitment of Moesin is required to retract polar blebs that allow cortical relaxation and dissipation of intracellular pressure. This fine balance of Moesin activity is further adjusted by Skittles and Pten, two enzymes that locally produce phosphoinositol 4,5-bisphosphate and thereby, regulate Moesin cortical association. These complementary pathways provide a spatiotemporal framework to explain how the cell cortex is remodeled throughout cell division.     

The stomatin/prohibitin/flotillin/HflK/C domain of flotillin-1 contains distinct sequences that direct plasma membrane localization and protein interactions in 3T3-L1 adipocytes

Flotillin-1 is a lipid raft-associated protein that has been implicated in various cellular processes. We examined the subcellular distribution of flotillin-1 in different cell types and found that localization is cell type-specific. Flotillin-1 relocates from a cytoplasmic compartment to the plasma membrane upon the differentiation of 3T3-L1 adipocytes. To delineate the structural determinants necessary for its localization, we generated a series of truncation mutants of flotillin-1. Wild type flotillin-1 has two putative hydrophobic domains and is localized to lipid raft microdomains at the plasma membrane. Flotillin-1 fragments lacking the N-terminal hydrophobic stretch are excluded from the lipid raft compartments but remain at the plasma membrane. On the other hand, mutants with the second hydrophobic region deleted fail to traffic to the plasma membrane but are instead found in intracellular granule-like structures. Flotillin-1 specifically interacts with the adaptor protein CAP, the Src family kinase Fyn, and cortical F-actin in lipid raft microdomains in adipocytes. Furthermore, CAP and Fyn associate with different regions in the N-terminal sequences of flotillin-1. These results furthered our understanding for how flotillin-1 can function as a molecular link between lipid rafts of the plasma membrane and a multimeric signaling complex at the actin cytoskeleton.     

Association of human immunodeficiency virus type 1 gag with membrane does not require highly basic sequences in the nucleocapsid: use of a novel Gag multimerization assay

Human immunodeficiency virus type 1 (HIV-1) particle production, a process driven by the Gag polyprotein precursor, occurs on the plasma membrane in most cell types. The plasma membrane contains cholesterol-enriched microdomains termed lipid rafts, which can be isolated as detergent-resistant membrane (DRM). Previously, we and others demonstrated that HIV-1 Gag is associated with DRM and that disruption of Gag-raft interactions impairs HIV-1 particle production. However, the determinants of Gag-raft association remain undefined. In this study, we developed a novel epitope-based Gag multimerization assay to examine whether Gag assembly is essential for its association with lipid rafts. We observed that membrane-associated, full-length Gag is poorly detected by immunoprecipitation relative to non-membrane-bound Gag. This poor detection is due to assembly-driven masking of Gag epitopes, as denaturation greatly improves immunoprecipitation. Gag mutants lacking the Gag-Gag interaction domain located in the N terminus of the nucleocapsid (NC) were efficiently immunoprecipitated without denaturation, indicating that the epitope masking is caused by higher-order Gag multimerization. We used this assay to examine the relationship between Gag assembly and Gag binding to total cellular membrane and DRM. Importantly, a multimerization-defective NC mutant displayed wild-type levels of membrane binding and DRM association, indicating that NC-mediated Gag multimerization is dispensable for association of Gag with membrane or DRM. We also demonstrate that different properties of sucrose and iodixanol membrane flotation gradients may explain some discrepancies regarding Gag-raft interactions. This report offers new insights into the association of HIV-1 Gag with membrane and with lipid rafts.     

Raft partitioning of the yeast uracil permease during trafficking along the endocytic pathway

Lipid rafts, formed by the lateral association of sphingolipids and cholesterol in the external membrane leaflet, have been implicated in membrane traffic and cell signaling in mammalian cells. Yeast plasma membranes were also recently shown to contain lipid raft microdomains consisting of sphingolipids and ergosterol, and containing several plasma membrane proteins, including Gas1p, a GPI-anchored protein, and the [H⁺] ATPase Pma1p. In this study, we investigated whether lipid rafts were involved in the intracellular trafficking of a yeast transporter, uracil permease, which undergoes ubiquitin-dependent endocytosis. Regardless of its ubiquitination status, uracil permease was found to be associated with rafts in the plasma membrane. The expression of Fur4p in lcb1–100 cells, deficient in the first enzyme of sphingolipid synthesis, impaired the association of Fur4p with detergent-resistant fractions. When targeted to endocytic compartments, uracil permease appeared to be progressively transferred to detergent-soluble fractions, suggesting that the lipid environment might change between plasma membrane and endosomes. Consistent with this hypothesis, the wild-type form of the v-SNARE Snc1p, which is known to cycle between the plasma membrane and endosomal compartments, was recovered in both detergent-resistant and detergent-soluble fractions. In contrast, a variant Snc1p that accumulates at the plasma membrane was recovered exclusively in detergent-resistant fractions .     

Reversible phosphorylation--dephosphorylation determines the localization of rab4 during the cell cycle.

The ras-like GTP binding protein rab4 is the only known rab protein on endosomes that is phosphorylated during mitosis. Since a large fraction of rab4 accumulates in the cytosol in mitotic cells, we investigated the molecular mechanism controlling membrane association of rab4. We first show that human rab4 is phosphorylated by recombinant mammalian p34cdc2 kinase in vitro. Next, the actual site of phosphorylation and its functional significance were determined using stably transfected CHO cell lines producing high levels of wild type rab4 or rab4 mutants bearing alterations at Ser196, which occurs within a consensus site for p34cdc2 kinase phosphorylation (S196PRR). Mutation of Ser196 to glutamine or aspartic acid completely prevented rab4 phosphorylation in mitotic cells and also blocked its appearance in the cytosol. Neither C-terminal isoprenylation nor carboxymethylation of rab4 was affected by the mutations or by phosphorylation. Finally, dephosphorylation and reassociation of soluble rab4 with membranes occurred upon exit of cells from mitosis. Thus, phosphorylation of Ser196 is directly responsible for the reversible translocation of rab4 into the cytosol of mitotic cells.     

A lipid-signaled myosin phosphatase surge disperses cortical contractile force early in cell spreading

When cells cease migrating through the vasculature, adhere to extracellular matrix, and begin to spread, they exhibit rapid changes in contraction and relaxation at peripheral regions newly contacting the underlying substrata. We describe here a requirement in this process for myosin II disassembly at the cell cortex via the action of myosin phosphatase (MP), which in turn is regulated by a plasma membrane signaling lipid. Cells in suspension exhibit high levels of activity of the signaling enzyme phospholipase D2 (PLD2), elevating production of the lipid second messenger phosphatidic acid (PA) at the plasma membrane, which in turn recruits MP and stores it there in a presumed inactive state. On cell attachment, down-regulation of PLD2 activity decreases PA production, leading to MP release, myosin dephosphorylation, and actomyosin disassembly. This novel model for recruitment and restraint of MP provides a means to effect a rapid cytoskeletal reorganization at the cell cortex upon demand.     

Localization of Pom121 to the inner nuclear membrane is required for an early step of interphase nuclear pore complex assembly

The nuclear pore complex (NPC) is a large protein assembly that mediates molecular trafficking between the cytoplasm and the nucleus. NPCs assemble twice during the cell cycle in metazoans: postmitosis and during interphase. In this study, using small interfering RNA (siRNA) in conjunction with a cell fusion-based NPC assembly assay, we demonstrated that pore membrane protein (Pom)121, a vertebrate-specific integral membrane nucleoporin, is indispensable for an early step in interphase NPC assembly. Functional domain analysis of Pom121 showed that its nuclear localization signals, which bind to importin β via importin α and likely function with RanGTP, play an essential role in targeting Pom121 to the interphase NPC. Furthermore, a region of Pom121 that interacts with the inner nuclear membrane (INM) and lamin B receptor was found to be crucial for its NPC targeting. Based on these findings and on evidence that Pom121 localizes at the INM in the absence of a complete NPC structure, we propose that the nuclear migration of Pom121 and its subsequent interaction with INM proteins are required to initiate interphase NPC assembly. Our data also suggest, for the first time, the importance of the INM as a seeding site for "prepores" during interphase NPC assembly.     

GPR37 Protein Trafficking to the Plasma Membrane Regulated by Prosaposin and GM1 Gangliosides Promotes Cell Viability

The subcellular distribution of the G protein-coupled receptor GPR37 affects cell viability and is implicated in the pathogenesis of parkinsonism. Intracellular accumulation and aggregation of GPR37 cause cell death, whereas GPR37 located in the plasma membrane provides cell protection. We define here a pathway through which the recently identified natural ligand, prosaposin, promotes plasma membrane association of GPR37. Immunoabsorption of extracellular prosaposin reduced GPR37^tGFP surface density and decreased cell viability in catecholaminergic N2a cells. We found that GPR37^tGFP partitioned in GM1 ganglioside-containing lipid rafts in the plasma membrane of live cells. This partitioning required extracellular prosaposin and was disrupted by lipid raft perturbation using methyl-β-cyclodextrin or cholesterol oxidase. Moreover, complex formation between GPR37^tGFP and the GM1 marker cholera toxin was observed in the plasma membrane. These data show functional association between GPR37, prosaposin, and GM1 in the plasma membrane. These results thus tie together the three previously defined components of the cellular response to insult. Our findings identify a mechanism through which the receptor''s natural ligand and GM1 may protect against toxic intracellular GPR37 aggregates observed in parkinsonism.