Fission Yeast Nod1 Is a Component of Cortical Nodes Involved in Cell Size Control and Division Site Placement

Most cells enter mitosis once they have reached a defined size. In the fission yeast Schizosaccharomyces pombe, mitotic entry is orchestrated by a geometry-sensing mechanism that involves the Cdk1/Cdc2-inhibiting Wee1 kinase. The factors upstream of Wee1 gather together in interphase to form a characteristic medial and cortical belt of nodes. Nodes are also considered to be precursors of the cytokinesis contractile actomyosin ring (CAR). Here we describe a new component of the interphase nodes and cytokinesis rings, which we named Nod1. Consistent with its role in cell size control at division, nod1Δ cells were elongated and epistatic with regulators of Wee1. Through biochemical and localisation studies, we placed Nod1 in a complex with the Rho-guanine nucleotide exchange factor Gef2. Nod1 and Gef2 mutually recruited each other in nodes and Nod1 also assembles Gef2 in rings. Like gef2Δ, nod1Δ cells showed a mild displacement of their division plane and this phenotype was severely exacerbated when the parallel Polo kinase pathway was also compromised. We conclude that Nod1 specifies the division site by localising Gef2 to the mitotic cell middle. Previous work showed that Gef2 in turn anchors factors that control the spatio-temporal recruitment of the actin nucleation machinery. It is believed that the actin filaments originated from the nodes pull nodes together into a single contractile ring. Surprisingly however, we found that node proteins could form pre-ring helical filaments in a cdc12-112 mutant in which nucleation of the actin ring is impaired. Furthermore, the deletion of either nod1 or gef2 created an un-expected situation where different ring components were recruited sequentially rather than simultaneously. At later stages of cytokinesis, these various rings appeared inter-fitted rather than merged. This study brings a new slant to the understanding of CAR assembly and function.     

A Highlights from MBoC Selection: Inhibition of cell membrane ingression at the division site by cell walls in fission yeast

Eukaryotic cells assemble actomyosin rings during cytokinesis to function as force-generating machines to drive membrane invagination and to counteract the intracellular pressure and the cell surface tension. How the extracellular matrix affects actomyosin ring contraction has not been fully explored. While studying the Schizosaccharomyces pombe 1,3-β-glucan-synthase mutant cps1-191, which is defective in division septum synthesis and arrests with a stable actomyosin ring, we found that weakening of the extracellular glycan matrix caused the generated spheroplasts to divide under the nonpermissive condition. This nonmedial slow division was dependent on a functional actomyosin ring and vesicular trafficking, but independent of normal septum synthesis. Interestingly, the high intracellular turgor pressure appears to play a minimal role in inhibiting ring contraction in the absence of cell wall remodeling in cps1-191 mutants, as decreasing the turgor pressure alone did not enable spheroplast division. We propose that during cytokinesis, the extracellular glycan matrix restricts actomyosin ring contraction and membrane ingression, and remodeling of the extracellular components through division septum synthesis relieves the inhibition and facilitates actomyosin ring contraction.     

Nuc2p, a subunit of the anaphase-promoting complex, inhibits septation initiation network following cytokinesis in fission yeast

In most cell types, mitosis and cytokinesis are tightly coupled such that cytokinesis occurs only once per cell cycle. The fission yeast Schizosaccharomyces pombe divides using an actomyosin-based contractile ring and is an attractive model for the study of the links between mitosis and cytokinesis. In fission yeast, the anaphase-promoting complex/cyclosome (APC/C) and the septation initiation network (SIN), a spindle pole body (SPB)–associated GTPase-driven signaling cascade, function sequentially to ensure proper coordination of mitosis and cytokinesis. Here, we find a novel interplay between the tetratricopeptide repeat (TPR) domain–containing subunit of the APC/C, Nuc2p, and the SIN, that appears to not involve other subunits of the APC/C. Overproduction of Nuc2p led to an increase in the presence of multinucleated cells, which correlated with a defect in actomyosin ring maintenance and localization of the SIN component protein kinases Cdc7p and Sid1p to the SPBs, indicative of defective SIN signaling. Conversely, loss of Nuc2p function led to increased SIN signaling, characterized by the persistent localization of Cdc7p and Sid1p on SPBs and assembly of multiple actomyosin rings and division septa. Nuc2p appears to function independently of the checkpoint with FHA and ring finger (CHFR)–related protein Dma1p, a known inhibitor of the SIN in fission yeast. Genetic and biochemical analyses established that Nuc2p might influence the nucleotide state of Spg1p GTPase, a key regulator of the SIN. We propose that Nuc2p, by inhibiting the SIN after cell division, prevents further deleterious cytokinetic events, thereby contributing to genome stability.     

Molecular organization of cytokinesis node predicts the constriction rate of the contractile ring

The molecular organization of cytokinesis proteins governs contractile ring function. We used single molecule localization microscopy in live cells to elucidate the molecular organization of cytokinesis proteins and relate it to the constriction rate of the contractile ring. Wild-type fission yeast cells assemble contractile rings by the coalescence of cortical proteins complexes called nodes whereas cells without Anillin/Mid1p (Δmid1) lack visible nodes yet assemble contractile rings competent for constriction from the looping of strands. We leveraged the Δmid1 contractile ring assembly mechanism to determine how two distinct molecular organizations, nodes versus strands, can yield functional contractile rings. Contrary to previous interpretations, nodes assemble in Δmid1 cells. Our results suggest that Myo2p heads condense upon interaction with actin filaments and an excess number of Myo2p heads bound to actin filaments hinders constriction thus reducing the constriction rate. Our work establishes a predictive correlation between the molecular organization of nodes and the behavior of the contractile ring.     

Involvement of an Actomyosin Contractile Ring in Saccharomyces cerevisiae Cytokinesis

In Saccharomyces cerevisiae, the mother cell and bud are connected by a narrow neck. The mechanism by which this neck is closed during cytokinesis has been unclear. Here we report on the role of a contractile actomyosin ring in this process. Myo1p (the only type II myosin in S. cerevisiae) forms a ring at the presumptive bud site shortly before bud emergence. Myo1p ring formation depends on the septins but not on F-actin, and preexisting Myo1p rings are stable when F-actin is depolymerized. The Myo1p ring remains in the mother–bud neck until the end of anaphase, when a ring of F-actin forms in association with it. The actomyosin ring then contracts to a point and disappears. In the absence of F-actin, the Myo1p ring does not contract. After ring contraction, cortical actin patches congregate at the mother–bud neck, and septum formation and cell separation rapidly ensue. Strains deleted for MYO1 are viable; they fail to form the actin ring but show apparently normal congregation of actin patches at the neck. Some myo1Δ strains divide nearly as efficiently as wild type; other myo1Δ strains divide less efficiently, but it is unclear whether the primary defect is in cytokinesis, septum formation, or cell separation. Even cells lacking F-actin can divide, although in this case division is considerably delayed. Thus, the contractile actomyosin ring is not essential for cytokinesis in S. cerevisiae. In its absence, cytokinesis can still be completed by a process (possibly localized cell–wall synthesis leading to septum formation) that appears to require septin function and to be facilitated by F-actin.     

Mechanisms of contractile-ring assembly in fission yeast and beyond

Most eukaryotes including fungi, amoebas, and animal cells assemble an actin/myosin-based contractile ring during cytokinesis. The majority of proteins implied in ring formation, maturation, and constriction are evolutionarily conserved, suggesting that common mechanisms exist among these divergent eukaryotes. Here, we review the recent advances in positioning and assembly of the actomyosin ring in the fission yeast Schizosaccharomyces pombe, the budding yeast Saccharomyces cerevisiae, and animal cells. In particular, major findings have been made recently in understanding ring formation in genetically tractable S. pombe, revealing a dynamic and robust search, capture, pull, and release mechanism.     

Nonmedially assembled F-actin cables incorporate into the actomyosin ring in fission yeast

In many eukaryotes, cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring. Despite the central role of this ring in cytokinesis, the mechanism of F-actin assembly and accumulation in the ring is not fully understood. In this paper, we investigate the mechanism of F-actin assembly during cytokinesis in Schizosaccharomyces pombe using lifeact as a probe to monitor actin dynamics. Previous work has shown that F-actin in the actomyosin ring is assembled de novo at the division site. Surprisingly, we find that a significant fraction of F-actin in the ring was recruited from formin-Cdc12p nucleated long actin cables that were generated at multiple nonmedial locations and incorporated into the ring by a combination of myosin II and myosin V activities. Our results, together with findings in animal cells, suggest that de novo F-actin assembly at the division site and directed transport of F-actin cables assembled elsewhere can contribute to ring assembly.     

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.     

An InCytes from MBC Selection: Roles of Formin Nodes and Myosin Motor Activity in Mid1p-dependent Contractile-Ring Assembly during Fission Yeast Cytokinesis

Two prevailing models have emerged to explain the mechanism of contractile-ring assembly during cytokinesis in the fission yeast Schizosaccharomyces pombe: the spot/leading cable model and the search, capture, pull, and release (SCPR) model. We tested some of the basic assumptions of the two models. Monte Carlo simulations of the SCPR model require that the formin Cdc12p is present in >30 nodes from which actin filaments are nucleated and captured by myosin-II in neighboring nodes. The force produced by myosin motors pulls the nodes together to form a compact contractile ring. Live microscopy of cells expressing Cdc12p fluorescent fusion proteins shows for the first time that Cdc12p localizes to a broad band of 30–50 dynamic nodes, where actin filaments are nucleated in random directions. The proposed progenitor spot, essential for the spot/leading cable model, usually disappears without nucleating actin filaments. α-Actinin ain1 deletion cells form a normal contractile ring through nodes in the absence of the spot. Myosin motor activity is required to condense the nodes into a contractile ring, based on slower or absent node condensation in myo2-E1 and UCS rng3-65 mutants. Taken together, these data provide strong support for the SCPR model of contractile-ring formation in cytokinesis.     

Glutamine Substitution at Alanine1649 in the S4–S5 Cytoplasmic Loop of Domain 4 Removes the Voltage Sensitivity of Fast Inactivation in the Human Heart Sodium Channel

Normal activation–inactivation coupling in sodium channels insures that inactivation is slow at small but rapid at large depolarizations. M1651Q/M1652Q substitutions in the cytoplasmic loop connecting the fourth and fifth transmembrane segments of Domain 4 (S4–S5/D4) of the human heart sodium channel subtype 1 (hH1) affect the kinetics and voltage dependence of inactivation (Tang, L., R.G. Kallen, and R. Horn. 1996. J. Gen. Physiol. 108:89–104.). We now show that glutamine substitutions NH₂-terminal to the methionines (L¹⁶⁴⁶, L¹⁶⁴⁷, F¹⁶⁴⁸, A¹⁶⁴⁹, L¹⁶⁵⁰) also influence the kinetics and voltage dependence of inactivation compared with the wild-type channel. In contrast, mutations at the COOH-terminal end of the S4–S5/D4 segment (L¹⁶⁵⁴, P¹⁶⁵⁵, A¹⁶⁵⁶) are without significant effect. Strikingly, the A1649Q mutation renders the current decay time constants virtually voltage independent and decreases the voltage dependences of steady state inactivation and the time constants for the recovery from inactivation. Single-channel measurements show that at negative voltages latency times to first opening are shorter and less voltage dependent in A1649Q than in wild-type channels; peak open probabilities are significantly smaller and the mean open times are shorter. This indicates that the rate constants for inactivation and, probably, activation are increased at negative voltages by the A1649Q mutation reminiscent of Y1494Q/ Y1495Q mutations in the cytoplasmic loop between the third and fourth domains (O''Leary, M.E., L.Q. Chen, R.G. Kallen, and R. Horn. 1995. J. Gen. Physiol. 106:641–658.). Other substitutions, A1649S and A1649V, decrease but fail to eliminate the voltage dependence of time constants for inactivation, suggesting that the decreased hydrophobicity of glutamine at either residues A¹⁶⁴⁹ or Y¹⁴⁹⁴Y¹⁴⁹⁵ may disrupt a linkage between S4–S5/D4 and the interdomain 3–4 loop interfering with normal activation–inactivation coupling.     

Regiospecificity of Dioxygenation of Di- to Pentachlorobiphenyls and Their Degradation to Chlorobenzoates by the bph-Encoded Catabolic Pathway of Burkholderia sp. Strain LB400

Burkholderia sp. strain LB400 is one of the most potent aerobic polychlorobiphenyl (PCB)-degrading microorganisms that have been characterized. Its PCB-dioxygenating activity originates predominantly or exclusively from the biphenyl dioxygenase encoded by its bph gene cluster. Analysis of the dioxygenation products of several di- to pentachlorinated biphenyls formed by this enzyme revealed a complex dependence of the regiospecificity and the yield of dioxygenation on the substitution patterns of both the oxidized and the nonoxidized rings. No dioxygenolytic attack involving chlorinated meta or para carbons was observed. Therefore, the ability of the enzyme to hydroxylate chlorinated carbons appears to be limited to the ortho position. However, it is not limited to monochlorinated rings, as evidenced by dioxygenation of the 2,4-disubstituted ring at carbons 2 and 3. This site of attack is strikingly different from that of the 2,5-dichlorinated ring, which has been shown to be dihydroxylated at positions 3 and 4 (J. D. Haddock, J. R. Horton, and D. T. Gibson, J. Bacteriol. 177:20–26, 1995). These results demonstrate that a second substituent of ortho-chlorinated rings crucially influences the site of dioxygenation at this ring and thereby determines whether or not the initial chlorobiphenyl oxidation product is further metabolized through the bph-encoded pathway. The 2,4-dichlorinated ring can alternatively be attacked at carbons 5 and 6. The preferred site crucially depends on the substitution pattern of the other ring. The formation of more than a single dioxygenation product was found predominantly with congeners that contain two chlorinated rings, both of which are similarly prone to dioxygenation or one is substituted only at carbon 3.     

Characterization of the roles of Blt1p in fission yeast cytokinesis

Spatial and temporal regulation of cytokinesis is essential for cell division, yet the mechanisms that control the formation and constriction of the contractile ring are incompletely understood. In the fission yeast Schizosaccharomyces pombe proteins that contribute to the cytokinetic contractile ring accumulate during interphase in nodes—precursor structures around the equatorial cortex. During mitosis, additional proteins join these nodes, which condense to form the contractile ring. The cytokinesis protein Blt1p is unique in being present continuously in nodes from early interphase through to the contractile ring until cell separation. Blt1p was shown to stabilize interphase nodes, but its functions later in mitosis were unclear. We use analytical ultracentrifugation to show that purified Blt1p is a tetramer. We find that Blt1p interacts physically with Sid2p and Mob1p, a protein kinase complex of the septation initiation network, and confirm known interactions with F-BAR protein Cdc15p. Contractile rings assemble normally in blt1∆ cells, but the initiation of ring constriction and completion of cell division are delayed. We find three defects that likely contribute to this delay. Without Blt1p, contractile rings recruited and retained less Sid2p/Mob1p and Clp1p phosphatase, and β-glucan synthase Bgs1p accumulated slowly at the cleavage site.     

Kinetic Characterization of the ATPase and Actin-activated ATPase Activities of Acanthamoeba castellanii Myosin-2

Phosphorylation of Ser-639 in loop-2 of the catalytic motor domain of the heavy chain of Acanthamoeba castellanii myosin-2 and the phosphomimetic mutation S639D have been shown previously to down-regulate the actin-activated ATPase activity of both the full-length myosin and single-headed subfragment-1 (Liu, X., Lee, D. Y., Cai, S., Yu, S., Shu, S., Levine, R. L., and Korn, E. D. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, E23–E32). In the present study we determined the kinetic constants for each step in the myosin and actomyosin ATPase cycles of recombinant wild-type S1 and S1-S639D. The kinetic parameter predominantly affected by the S639D mutation is the actin-activated release of inorganic phosphate from the acto myosin·ADP·P_i complex, which is the rate-limiting step in the steady-state actomyosin ATPase cycle. As consequence of this change, the duty ratio of this conventional myosin decreases. We speculate on the effect of Ser-639 phosphorylation on the processive behavior of myosin-2 filaments.     

A Lethal de Novo Mutation in the Middle Domain of the Dynamin-related GTPase Drp1 Impairs Higher Order Assembly and Mitochondrial Division

Mitochondria dynamically fuse and divide within cells, and the proper balance of fusion and fission is necessary for normal mitochondrial function, morphology, and distribution. Drp1 is a dynamin-related GTPase required for mitochondrial fission in mammalian cells. It harbors four distinct domains: GTP-binding, middle, insert B, and GTPase effector. A lethal mutation (A395D) within the Drp1 middle domain was reported in a neonate with microcephaly, abnormal brain development, optic atrophy, and lactic acidemia (Waterham, H. R., Koster, J., van Roermund, C. W., Mooyer, P. A., Wanders, R. J., and Leonard, J. V. (2007) N. Engl. J. Med. 356, 1736–1741). Mitochondria within patient-derived fibroblasts were markedly elongated, but the molecular mechanisms underlying these findings were not demonstrated. Because the middle domain is particularly important for the self-assembly of some dynamin superfamily proteins, we tested the hypothesis that this A395D mutation, and two other middle domain mutations (G350D, G363D) were important for Drp1 tetramerization, higher order assembly, and function. Although tetramerization appeared largely intact, each of these mutations compromised higher order assembly and assembly-dependent stimulation of Drp1 GTPase activity. Moreover, mutant Drp1 proteins exhibited impaired localization to mitochondria, indicating that this higher order assembly is important for mitochondrial recruitment, retention, or both. Overexpression of these middle domain mutants markedly inhibited mitochondrial division in cells. Thus, the Drp1 A395D lethal defect likely resulted in impaired higher order assembly of Drp1 at mitochondria, leading to decreased fission, elongated mitochondria, and altered cellular distribution of mitochondria.     

Three-dimensional Patterns and Redistribution of Myosin II and Actin in Mitotic Dictyostelium Cells

Myosin II is not essential for cytokinesis in cells of Dictyostelium discoideum that are anchored on a substrate (Neujahr, R., C. Heizer, and G. Gerisch. 1997. J. Cell Sci. 110:123–137), in contrast to its importance for cell division in suspension (DeLozanne, A., and J.A. Spudich. 1987. Science. 236:1086–1091; Knecht, D.A., and W.F. Loomis. 1987. Science. 236: 1081–1085.). These differences have prompted us to investigate the three-dimensional distribution of myosin II in cells dividing under one of three conditions: (a) in shaken suspension, (b) in a fluid layer on a solid substrate surface, and (c) under mechanical stress applied by compressing the cells. Under the first and second conditions outlined above, myosin II does not form patterns that suggest a contractile ring is established in the furrow. Most of the myosin II is concentrated in the regions that flank the furrow on both sides towards the poles of the dividing cell. It is only when cells are compressed that myosin II extensively accumulates in the cleavage furrow, as has been previously described (Fukui, Y., T.J. Lynch, H. Brzeska, and E.D. Korn. 1989. Nature. 341:328–331), i.e., this massive accumulation is a response to the mechanical stress. Evidence is provided that the stress-associated translocation of myosin II to the cell cortex is a result of the dephosphorylation of its heavy chains. F-actin is localized in the dividing cells in a distinctly different pattern from that of myosin II. The F-actin is shown to accumulate primarily in protrusions at the two poles that ultimately form the leading edges of the daughter cells. This distribution changes dynamically as visualized in living cells with a green fluorescent protein–actin fusion.     

Brush Border Myosin–I Structure and ADP-dependent Conformational Changes Revealed by Cryoelectron Microscopy and Image Analysis

Brush border myosin–I (BBM-I) is a single-headed myosin found in the microvilli of intestinal epithelial cells, where it forms lateral bridges connecting the core bundle of actin filaments to the plasma membrane. Extending previous observations (Jontes, J.D., E.M. Wilson-Kubalek, and R.A. Milligan. 1995. Nature [Lond.]. 378:751–753), we have used cryoelectron microscopy and helical image analysis to generate three-dimensional (3D) maps of actin filaments decorated with BBM-I in both the presence and absence of 1 mM MgADP. In the improved 3D maps, we are able to see the entire light chain–binding domain, containing density for all three calmodulin light chains. This has enabled us to model a high resolution structure of BBM-I using the crystal structures of the chicken skeletal muscle myosin catalytic domain and essential light chain. Thus, we are able to directly measure the full magnitude of the ADP-dependent tail swing. The ~31° swing corresponds to ~63 Å at the end of the rigid light chain–binding domain. Comparison of the behavior of BBM-I with skeletal and smooth muscle subfragments-1 suggests that there are substantial differences in the structure and energetics of the biochemical transitions in the actomyosin ATPase cycle.     

Differences in Human Cortical Gene Expression Match the Temporal Properties of Large-Scale Functional Networks

We explore the relationships between the cortex functional organization and genetic expression (as provided by the Allen Human Brain Atlas). Previous work suggests that functional cortical networks (resting state and task based) are organized as two large networks (differentiated by their preferred information processing mode) shaped like two rings. The first ring–Visual-Sensorimotor-Auditory (VSA)–comprises visual, auditory, somatosensory, and motor cortices that process real time world interactions. The second ring–Parieto-Temporo-Frontal (PTF)–comprises parietal, temporal, and frontal regions with networks dedicated to cognitive functions, emotions, biological needs, and internally driven rhythms. We found–with correspondence analysis–that the patterns of expression of the 938 genes most differentially expressed across the cortex organized the cortex into two sets of regions that match the two rings. We confirmed this result using discriminant correspondence analysis by showing that the genetic profiles of cortical regions can reliably predict to what ring these regions belong. We found that several of the proteins–coded by genes that most differentiate the rings–were involved in neuronal information processing such as ionic channels and neurotransmitter release. The systematic study of families of genes revealed specific proteins within families preferentially expressed in each ring. The results showed strong congruence between the preferential expression of subsets of genes, temporal properties of the proteins they code, and the preferred processing modes of the rings. Ionic channels and release-related proteins more expressed in the VSA ring favor temporal precision of fast evoked neural transmission (Sodium channels SCNA1, SCNB1 potassium channel KCNA1, calcium channel CACNA2D2, Synaptotagmin SYT2, Complexin CPLX1, Synaptobrevin VAMP1). Conversely, genes expressed in the PTF ring favor slower, sustained, or rhythmic activation (Sodium channels SCNA3, SCNB3, SCN9A potassium channels KCNF1, KCNG1) and facilitate spontaneous transmitter release (calcium channel CACNA1H, Synaptotagmins SYT5, Complexin CPLX3, and synaptobrevin VAMP2).     

Rho-mediated Contractility Exposes a Cryptic Site in Fibronectin and Induces Fibronectin Matrix Assembly

Many factors influence the assembly of fibronectin into an insoluble fibrillar extracellular matrix. Previous work demonstrated that one component in serum that promotes the assembly of fibronectin is lysophosphatidic acid (Zhang, Q., W.J. Checovich, D.M. Peters, R.M. Albrecht, and D.F. Mosher. 1994. J. Cell Biol. 127:1447–1459). Here we show that C3 transferase, an inhibitor of the low molecular weight GTP-binding protein Rho, blocks the binding of fibronectin and the 70-kD NH₂-terminal fibronectin fragment to cells and blocks the assembly of fibronectin into matrix induced by serum or lysophosphatidic acid. Microinjection of recombinant, constitutively active Rho into quiescent Swiss 3T3 cells promotes fibronectin matrix assembly by the injected cells. Investigating the mechanism by which Rho promotes fibronectin polymerization, we have used C3 to determine whether integrin activation is involved. Under conditions where C3 decreases fibronectin assembly we have only detected small changes in the state of integrin activation. However, several inhibitors of cellular contractility, that differ in their mode of action, inhibit cell binding of fibronectin and the 70-kD NH₂-terminal fibronectin fragment, decrease fibronectin incorporation into the deoxycholate insoluble matrix, and prevent fibronectin's assembly into fibrils on the cell surface. Because Rho stimulates contractility, these results suggest that Rho-mediated contractility promotes assembly of fibronectin into a fibrillar matrix. One mechanism by which contractility could enhance fibronectin assembly is by tension exposing cryptic self-assembly sites within fibronectin that is being stretched. Exploring this possibility, we have found a monoclonal antibody, L8, that stains fibronectin matrices differentially depending on the state of cell contractility. L8 was previously shown to inhibit fibronectin matrix assembly (Chernousov, M.A., A.I. Faerman, M.G. Frid, O.Y. Printseva, and V.E. Koteliansky. 1987. FEBS (Fed. Eur. Biochem. Soc.) Lett. 217:124–128). When it is used to stain normal cultures that are developing tension, it reveals a matrix indistinguishable from that revealed by polyclonal anti-fibronectin antibodies. However, the staining of fibronectin matrices by L8 is reduced relative to the polyclonal antibody when the contractility of cells is inhibited by C3. We have investigated the consequences of mechanically stretching fibronectin in the absence of cells. Applying a 30–35% stretch to immobilized fibronectin induced binding of soluble fibronectin, 70-kD fibronectin fragment, and L8 monoclonal antibody. Together, these results provide evidence that self-assembly sites within fibronectin are exposed by tension.     

Anillin-related protein Mid1p coordinates the assembly of the cytokinetic contractile ring in fission yeast

In fission yeast cells cortical nodes containing the protein Blt1p and several kinases appear early in G2, mature into cytokinetic nodes by adding anillin Mid1p, myosin-II, formin Cdc12p, and other proteins, and condense into a contractile ring by movements that depend on actin and myosin-II. Previous studies concluded that cells without Mid1p lack cytokinetic nodes and assemble rings unreliably from myosin-II strands but left open questions. Why do strands form outside the equatorial region? Why is ring assembly unreliable without Mid1p? We found in Δmid1 cells that Cdc12p accumulates in cytokinetic nodes scattered in the cortex and produces actin filaments that associate with myosin-II, Rng2p, and Cdc15p to form strands located between the nodes. Strands incorporate nodes, and in ∼67% of cells, strands slowly close into rings that constrict without the normal ∼25-min maturation period. Ring assembly is unreliable and slow without Mid1p because the scattered Cdc12p nodes generate strands spread widely beyond the equator, and growing strands depend on random encounters to merge with other strands into a ring. We conclude that orderly assembly of the contractile ring in wild-type cells depends on Mid1p to recruit myosin-II, Rng2p, and Cdc15p to nodes and to place cytokinetic nodes around the cell equator.     

Modulation of the substrate binding sites of platelet myosin by actin.

The studies reported in this paper were undertaken to compare the steady-state kinetics of ATPase of purified platelet actomyosin and myosin free of actin. Actomyosin exhibits highly sigmoid kinetics with at least two interacting ATP or UTP binding sites. These studies were done at O.6 m KCl where actin and myosin are generally supposed to be dissociated in the presence of these nucleotides (M. Gallaghar, T. C. Detwiler, and A. Stracher, 1976, in Cell Motility (Goldman, R., Pollard, T., and Rosenbaum, J., eds.), Vol. 3, Part A, pp. 475–485 Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.; A. Weber, 1969, J. Gen Physiol.53, 781–791). When the dissociation of platelet actomyosin was actually investigated by a sucrose density gradient technique under conditions which were similar to those of the steady-state kinetic experiments, only partial dissociation of actin from myosin was observed. This was especially true at low nucleotide concentrations where differences in the sigmoidicity of the saturation curves of actomyosin and actin-free myosin have been observed. These findings suggest that in platelet actomyosin, actin enhances the cooperativity of nucleotide binding sites of myosin by reducing the K_m for ATP or UTP. In contrast, the saturation curves of platelet myosin using either ATP or UTP as substrates are less sigmoidal and possess an intermediary plateau region, when analyzed by Hill and reciprocal plots, these data indicate both positive and negative cooperativity suggesting more than two substrate binding sites. Platelet myosin also hydrolyzed other nucleotides (the order of rates being ITP > UTP > UTP > ATP > CTP > GTP). The kinetics of ITP differed from that of ATP or UTP in that no plateau region was observed on the saturation curve. In addition, no cooperativity of ITP binding sites was seen at low substrate concentrations (up to 0.2 mm) but was instead observed at high ITP concentration. It is concluded that conformational changes in myosin induced by ITP may not be necessarily identical to those induced by ATP or UTP.