Diphosphorylated MRLC is required for organization of stress fibers in interphase cells and the contractile ring in dividing cells

Activity of nonmuscle myosin II is regulated by phosphorylation of its regulatory light chain (MRLC). Phosphoryration of MRLC at both Thr18 and Ser19 (diphosphorylation) results in higher MgATPase activity and in promotion of the assembly of myosin II filaments than does that of MRLC at Ser19 (monophosphorylation) in vitro. To determine the roles of the diphosphorylated MRLC in vivo, we transfected three kinds of MRLC mutants, unphosphorylated, monophosphorylated and diphosphorylated forms (MRLC2(T18AS19A), substitution of both Ser19 and Thr18 by Ala; MRLC2(T18AS19D), Ser19 by Asp and Thr18 by Ala; and MRLC2(T18DS19D), both Ser19 and Thr18 by Asp, respectively), into HeLa cells. Cells overexpressing the mutant MRLC2(T18DS19D) contained a larger number of actin filament bundles than did those overexpressing the mutant MRLC2(T18AS19D). Moreover, cells overexpressing the nonphosphorylatable mutant MRLC2(T18AS19A) showed a decrease in the number of actin filament bundles. Taken together, our data suggest that diphosphorylation of MRLC plays an important role in regulating actin filament assembly and reorganization in nonmuscle cells.     

Membrane associated nonmuscle myosin II functions as a motor for actin-based vesicle transport in clam oocyte extracts

Nonmuscle myosin II (Myo2) has been shown to associate with membranes of the trans-Golgi network and to be involved in Golgi to ER retrograde protein transport. Here, we provide evidence that Myo2 not only associates with membranes but functions to transport vesicles on actin filaments (AFs). We used extracts from unactivated clam oocytes for these studies. AFs assembled spontaneously in these extracts and myosin-dependent vesicle transport was observed upon activation. In addition, actin bundles formed and moved relative to each other at an average speed of 0.30 microm/s. Motion analysis revealed that vesicles moved on the spontaneously assembled AFs at speeds greater than 1 microm/s. The motor on these vesicles was identified as a member of the nonmuscle Myo2 family based on sequence determination by Edman chemistry. Vesicles in these extracts were purified by sucrose gradient centrifugation and movement was reconstituted in vitro using skeletal muscle actin coated coverslips. When peripheral membrane proteins of vesicles including Myo2 were removed by salt stripping or when extracts were treated with an antibody specific to clam oocyte nonmuscle Myo2, vesicle movement was inhibited. Blebbistatin, a Myo2 specific inhibitor, also blocked vesicle movement. Myo2 light chain kinase activity was found to be essential for vesicle movement and sliding of actin bundles. Together, our data provide direct evidence that nonmuscle Myo2 is involved in actin-dependent vesicle transport in clam oocytes.     

Myosin is involved in postmitotic cell spreading

We have investigated a role for myosin in postmitotic Potoroo tridactylis kidney (PtK2) cell spreading by inhibitor studies, time- lapse video microscopy, and immunofluorescence. We have also determined the spatial organization and polarity of actin filaments in postmitotic spreading cells. We show that butanedione monoxime (BDM), a known inhibitor of muscle myosin II, inhibits nonmuscle myosin II and myosin V adenosine triphosphatases. BDM reversibly inhibits PtK2 postmitotic cell spreading. Listeria motility is not affected by this drug. Electron microscopy studies show that some actin filaments in spreading edges are part of actin bundles that are also found in long, thin, structures that are connected to spreading edges and substrate (retraction fibers), and that 90% of this actin is oriented with barbed ends in the direction of spreading. The remaining actin in spreading edges has a more random orientation and spatial arrangement. Myosin II is associated with actin polymer in spreading cell edges, but not retraction fibers. Myosin II is excluded from lamellipodia that protrude from the cell edge at the end of spreading. We suggest that spreading involves myosin, possibly myosin II.     

Cytoskeleton mediating transport between the ER system and the Golgi apparatus in the green alga Scenedesmus acutus

In the green alga Scenedesmus acutus, Golgi bodies are located near the nucleus and supplied with transition vesicles that bud from the outer nuclear envelope membrane. Using this alga, we have shown previously that thiamine pyrophosphatase (TPPase), a marker enzyme of Golgi bodies, migrates in vesicles from the Golgi bodies to the ER via the nuclear envelope in the presence of BFA (Noguchi et al., Protoplasma 201, 202-212, 1998). In this study we demonstrate that both cytochalasin B and oryzalin (microtubule-disrupting agent) inhibit the BFA-induced migration of TPPase from Golgi bodies to the nuclear envelope. However, only actin filaments--not microtubules--can be detected between the nuclear envelope and the Golgi bodies in both BFA-treated and untreated cells. These observations suggest that actin filaments mediate the BFA-induced retrograde transport of vesicles. This mechanism differs from that found in mammalian cells, in which microtubules mediate BFA-induced retrograde transport by the elongation of membrane tubules from the Golgi cisternae. We also discuss the non-participation of the cytoskeleton in anterograde transport from the nuclear envelope to the Golgi bodies.     

Brefeldin A redistributes resident and itinerant Golgi proteins to the endoplasmic reticulum

Brefeldin A (BFA) has been reported to block protein transport from the ER and cause disassembly of the Golgi complex. We have examined the effects of BFA on the transport and processing of the vesicular stomatitis virus G protein, a model integral membrane protein. Delivery of G protein to the cell surface was reversibly blocked by 6 micrograms/ml BFA. Pulse-label experiments revealed that in the presence of BFA, G protein became completely resistant to endoglycosidase H digestion. Addition of sialic acid, a trans-Golgi event, was not observed. Despite processing by cis- and medial Golgi enzymes, G protein was localized by indirect immunofluorescence to a reticular distribution characteristic of the ER. By preventing transport of G protein from the ER with the metabolic inhibitor carbonyl cyanide m-chlorophenylhydrazone or by use of the temperature-sensitive mutant ts045, which is restricted to the ER at 40 degrees C, we showed that processing of G protein occurred in the ER and was not due to retention of newly synthesized Golgi enzymes. Rather, redistribution of preexisting cis and medial Golgi enzymes to the ER occurred as soon as 2.5 min after addition of BFA, and was complete by 10-15 min. Delivery of Golgi enzymes to the ER was energy dependent and occurred only at temperatures greater than or equal to 20 degrees C. BFA also induced retrograde transport of G protein from the medial Golgi to the ER. Golgi enzymes were completely recovered from the ER 10 min after removal of BFA. These findings demonstrate that BFA induces retrograde transport of both resident and itinerant Golgi proteins to the ER in a fully reversible manner.     

Does 2,3-butanedione monoxime inhibit nonmuscle myosin?

Summary. BDM (2,3-butanedione monoxime) has been used extensively to inhibit nonmuscle myosin. However, recent articles raise the question of what BDM actually does, because of experiments in which BDM did not affect the actin-activated ATPase of nonmuscle myosins. We describe results that indicate that BDM indeed inhibits motility due to nonmuscle myosins: in many different cells BDM has the same effects as anti-actin agents and/or as other anti-myosin agents, and BDM slows or stops the sliding between actin filaments and myosin in vitro. We discuss how the two sets of apparently contradictory results might be resolved, and we suggest possible experiments that might clarify the contradictory interpretations. Correspondence and reprints: Biology Department, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.     

Myosin II Is Involved in the Production of Constitutive Transport Vesicles from the TGN

The participation of nonmuscle myosins in the transport of organelles and vesicular carriers along actin filaments has been documented. In contrast, there is no evidence for the involvement of myosins in the production of vesicles involved in membrane traffic. Here we show that the putative TGN coat protein p200 (Narula, N., I. McMorrow, G. Plopper, J. Doherty, K.S. Matlin, B. Burke, and J.L. Stow. 1992. J. Cell Biol. 114: 1113–1124) is myosin II. The recruitment of myosin II to Golgi membranes is dependent on actin and is regulated by G proteins. Using an assay that studies the release of transport vesicles from the TGN in vitro, we provide functional evidence that p200/myosin is involved in the assembly of basolateral transport vesicles carrying vesicular stomatitis virus G protein (VSVG) from the TGN of polarized MDCK cells. The 50% reduced efficiency in VSVG vesicle release from the TGN in vitro after depletion of p200/myosin II could be reestablished to control levels by the addition of purified nonmuscle myosin II. Several inhibitors of the actin-stimulated ATPase activity of myosin specifically inhibited the release of VSVG-containing vesicles from the TGN.     

Retrograde transport from the Golgi complex to the ER of both Shiga toxin and the nontoxic Shiga B-fragment is regulated by butyric acid and cAMP

Endocytosed Shiga toxin is transported from the Golgi complex to the endoplasmic reticulum in butyric acid-treated A431 cells. We here examine the extent of this retrograde transport and its regulation. The short B fragment of Shiga toxin is sufficient for transport to the ER. The B fragment of cholera toxin, which also binds to glycolipids, is transported to all the Golgi cisterns, but cannot be localized in the ER even after butyric acid treatment. Under all conditions the toxic protein ricin was found predominantly in the trans-Golgi network. There is no transport of endocytosed fluid to the Golgi apparatus or to the ER even after butyric acid treatment and in the presence of Shiga toxin, indicating that transport to the ER, through the trans-Golgi network and the cisterns of the Golgi apparatus, involves several sorting stations. Since Shiga toxin receptors (Gb3) in butyric acid- treated A431 cells seem to have a ceramide moiety with longer fatty acids than in untreated cells, the possibility exists that fatty acid composition of the receptor is important for sorting to the ER. Both retrograde transport and intoxication with Shiga toxin can also be induced by cAMP, supporting the idea that retrograde transport from the Golgi to the ER is required for intoxication. The data suggest that transport to the ER in cells in situ may depend on fatty acid composition and is regulated by physiological signals.     

Regulation of protein transport from the Golgi complex to the endoplasmic reticulum by CDC42 and N-WASP

Actin is involved in the organization of the Golgi complex and Golgi-to-ER protein transport in mammalian cells. Little, however, is known about the regulation of the Golgi-associated actin cytoskeleton. We provide evidence that Cdc42, a small GTPase that regulates actin dynamics, controls Golgi-to-ER protein transport. We located GFP-Cdc42 in the lateral portions of Golgi cisternae and in COPI-coated and non-coated Golgi-associated transport intermediates. Overexpression of Cdc42 and its activated form Cdc42V12 inhibited the retrograde transport of Shiga toxin from the Golgi complex to the ER, the redistribution of the KDEL receptor, and the ER accumulation of Golgi-resident proteins induced by the active GTP-bound mutant of Sar1 (Sar1[H79G]). Coexpression of wild-type or activated Cdc42 and N-WASP also inhibited Golgi-to-ER transport, but this was not the case in cells expressing Cdc42V12 and N-WASP(Delta WA), a mutant form of N-WASP that lacks Arp2/3 binding. Furthermore, Cdc42V12 recruited GFP-N-WASP to the Golgi complex. We therefore conclude that Cdc42 regulates Golgi-to-ER protein transport in an N-WASP-dependent manner.     

Regulation of myosin II dynamics by phosphorylation and dephosphorylation of its light chain in epithelial cells

Nonmuscle myosin II, an actin-based motor protein, plays an essential role in actin cytoskeleton organization and cellular motility. Although phosphorylation of its regulatory light chain (MRLC) is known to be involved in myosin II filament assembly and motor activity in vitro, it remains unclear exactly how MRLC phosphorylation regulates myosin II dynamics in vivo. We established clones of Madin Darby canine kidney II epithelial cells expressing MRLC-enhanced green fluorescent protein or its mutants. Time-lapse imaging revealed that both phosphorylation and dephosphorylation are required for proper dynamics of myosin II. Inhibitors affecting myosin phosphorylation and MRLC mutants indicated that monophosphorylation of MRLC is required and sufficient for maintenance of stress fibers. Diphosphorylated MRLC stabilized myosin II filaments and was distributed locally in regions of stress fibers where contraction occurs, suggesting that diphosphorylation is involved in the spatial regulation of myosin II assembly and contraction. We further found that myosin phosphatase or Zipper-interacting protein kinase localizes to stress fibers depending on the activity of myosin II ATPase.     

Redistribution of membrane proteins between the Golgi apparatus and endoplasmic reticulum in plants is reversible and not dependent on cytoskeletal networks

We have fused the signal anchor sequences of a rat sialyl transferase and a human galactosyl transferase along with the Arabidopsis homologue of the yeast HDEL receptor (AtERD2) to the jellyfish green fluorescent protein (GFP) and transiently expressed the chimeric genes in tobacco leaves. All constructs targeted the Golgi apparatus and co-expression with DsRed fusions along with immunolabelling of stably transformed BY2 cells indicated that the fusion proteins located all Golgi stacks. Exposure of tissue to brefeldin A (BFA) resulted in the reversible redistribution of ST-GFP into the endoplasmic reticulum. This effect occurred in the presence of a protein synthesis inhibitor and also in the absence of microtubules or actin filaments. Likewise, reformation of Golgi stacks on removal of BFA was not dependent on either protein synthesis or the cytoskeleton. These data suggest that ER to Golgi transport in the cell types observed does not require cytoskeletal-based mechanochemical motor systems. However, expression of an inhibitory mutant of Arabidopsis Rab 1b (AtRab1b(N121I) significantly slowed down the recovery of Golgi fluorescence in BFA treated cells indicating a role for Rab1 in regulating ER to Golgi anterograde transport.     

Actomyosin mediates gravisensing and early transduction events in reoriented cut snapdragon spikes

We investigated the involvement of the actomyosin network in the early events of the gravitropic response of cut snapdragon (Antirrhinum majus L.) spikes. The effects of the actin-modulating drug, cytochalasin D (CD) and/or the myosin inhibitor, 2,3-butanedione-2-monoxime (BDM) on amyloplast displacement, lateral auxin transport and consequently on stem bending were examined. The inhibitory effect on cytoskeleton integrity was studied by using indirect immunofluorescence double-labeling of actin and myosin. Our results demonstrate that no organizational changes in actin filaments occurred in cortical and endodermal cells of the stem bending zone during reorientation. These results suggest that actin depolymerization is not required for amyloplast sedimentation. Unlike the chloroplasts in the cortex, the amyloplasts in the endodermis were surrounded by actin and myosin, indicating that amyloplasts may be attached to the actin filaments via the motor protein, myosin. This suggests the involvement of myosin as part of the actomyosin complex in amyloplast movement in vertical as well as in reoriented stems. This suggestion was supported by the findings showing that: (a) BDM or CD disrupted the normal organization of actin either by altering characteristic distribution patterns of myosin-like protein in the cortex (BDM), or by causing actin fragmentation (CD); (b) both compounds inhibited the gravity-induced amyloplast displacement in the endodermis. Additionally, these compounds also inhibited lateral auxin transport across the stem and stem gravitropic bending. Our study suggests that during stem reorientation amyloplasts possibly remain attached to the actin filaments, using myosin as a motor protein. Thus, gravisensing and early transduction events in the gravitropic response of snapdragon spikes, manifested by amyloplast displacement and lateral auxin transport, are mediated by the actomyosin complex.     

Clofibrate inhibits membrane trafficking to the Golgi complex and induces its retrograde movement to the endoplasmic reticulum

Insights into the function of the Golgi complex have been provided by experiments performed with various inhibitors of membrane trafficking, such as the macrocyclic lactone brefeldin A (BFA), a compound that inhibits constitutive secretion, prevents the formation of coatomer-coated transport vesicles, and stimulates the retrograde movement of Golgi resident enzymes back to the ER. We show here that the structurally unrelated compound clofibrate, a peroxisome proliferator (PP) and hypolipidemic agent, also reversibly disrupts the morphological and functional integrity of the Golgi complex in a manner similar to BFA. In the presence of clofibrate, the forward transport of newly synthesized secretory proteins from the ER to the Golgi is dramatically inhibited. Moreover, clofibrate causes Golgi membranes to travel rapidly in a microtubule-dependent manner back to the ER, forming a hybrid ER–Golgi tubulovesicular membrane network. These affects appear to be independent of clofibrate's ability to stimulate the PP-activated receptor (PPAR) alpha pathway because other PPAR stimulators (DEHP, WY-14643) did not alter the Golgi complex or induce retrograde trafficking. These data suggest that PPAR alpha-independent, clofibrate-sensitive proteins participate in regulating Golgi-to-ER retrograde membrane transport, and, equally importantly, that clofibrate may be used as a pharmacological tool for investigating Golgi membrane dynamics.     

Electron Tomography and Simulation of Baculovirus Actin Comet Tails Support a Tethered Filament Model of Pathogen Propulsion

Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion, the underlying mechanism of pathogen movement is currently speculative and controversial. Using electron tomography we have resolved the three-dimensional architecture of actin comet tails propelling baculovirus, the smallest pathogen yet known to hijack the actin motile machinery. Comet tail geometry was also mimicked in mixtures of virus capsids with purified actin and a minimal inventory of actin regulators. We demonstrate that propulsion is based on the assembly of a fishbone-like array of actin filaments organized in subsets linked by branch junctions, with an average of four filaments pushing the virus at any one time. Using an energy-minimizing function we have simulated the structure of actin comet tails as well as the tracks adopted by baculovirus in infected cells in vivo. The results from the simulations rule out gel squeezing models of propulsion and support those in which actin filaments are continuously tethered during branch nucleation and polymerization. Since Listeria monocytogenes, Shigella flexneri, and Vaccinia virus among other pathogens use the same common toolbox of components as baculovirus to move, we suggest they share the same principles of actin organization and mode of propulsion.     

Uncoupling of brefeldin a-mediated coatomer protein complex-I dissociation from Golgi redistribution

The Golgi complex functions in transport of molecules from the endoplasmic reticulum (ER) to the plasma membrane and other distal organelles as well as in retrograde transport to the ER. The fungal metabolite brefeldin A (BFA) promotes dissociation of ADP-ribosylation-factor-1 (ARF1) and the coatomer protein complex-I (COP-I) from Golgi membranes, followed by Golgi tubulation and fusion with the ER. Here we demonstrate that the cationic ionophore monensin inhibited the BFA-mediated Golgi redistribution to the ER without interfering with ARF1 and COP-I dissociation. Preservation of a perinuclear Golgi despite COP-I and ARF1 dissociation enables addressing the involvement of these proteins in anterograde ER to Golgi transport. The thermo-reversible folding mutant of vesicular stomatitis virus G protein (VSVGtsO45) was retained in the ER in the presence of both monensin and BFA, thus supporting ARF1/COP-I participation in ER-exit processes. Live-cell imaging revealed that BFA-induced Golgi tubulation persisted longer in the presence of monensin, suggesting that monensin inhibits tubule fusion with the ER. Moreover, monensin also augmented Golgi-derived tubules that contained the ER-Golgi-intermediate compartment marker, p58, in the absence of BFA, signifying the generality of this effect. Taken together, we propose that monensin inhibits membrane fusion processes in the presence or absence of BFA.     

Effects of brefeldin A on the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum in a green alga,Scenedesmus acutus

Summary The effects of brefeldin A (BFA) on the structure of the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum (ER), and on the thiamine pyrophosphatase (TPPase) activity in these organelles were examined in a green alga,Scenedesmus acutus, to obtain evidence for the existence of a retrograde transport from the Golgi apparatus to the ER via the nuclear envelope. InScenedesmus, Golgi bodies are situated close to the nuclear envelope throughout the cell cycle and receive the transition vesicles not directly from the ER, but from the nuclear envelope. BFA induced the disassembly of Golgi bodies and an increase in the ER cisternae at the trans-side of decomposed Golgi bodies in interphase cells and multinuclear cells before septum formation. The accumulated ER cisternae connected to the nuclear envelope at one part. TPPase activity was detected in all cisternae of Golgi bodies, but not in the nuclear envelope or the ER in nontreated cells. On the contrary, in BFA-treated cells, TPPase activity was detected in the nuclear envelope and the ER in addition to the decomposed Golgi bodies. When septum-forming cells were treated with BFA, the disassembly of Golgi bodies was less than that in interphase cells, and TPPase activity was detected in the Golgi cisternae but not in the nuclear envelope or the ER. These results suggest mat BFA blocks the anterograde transport from the nuclear envelope to the Golgi bodies but does not block the retrograde transport from the Golgi bodies to the nuclear envelope in interphase and multinuclear cells.Abbreviations BFA brefeldin A - ER endoplasmic reticulum - TPPase thiamine pyrophosphatase     

The secretion inhibitor Exo2 perturbs trafficking of Shiga toxin between endosomes and the trans-Golgi network

The small-molecule inhibitor Exo2 {4-hydroxy-3-methoxy-(5,6,7,8-tetrahydrol[1]benzothieno[2,3-d]pyrimidin-4-yl)hydraz-one benzaldehyde} has been reported to disrupt the Golgi apparatus completely and to stimulate Golgi-ER (endoplasmic reticulum) fusion in mammalian cells, akin to the well-characterized fungal toxin BFA (brefeldin A). It has also been reported that Exo2 does not affect the integrity of the TGN (trans-Golgi network), or the direct retrograde trafficking of the glycolipid-binding cholera toxin from the TGN to the ER lumen. We have examined the effects of BFA and Exo2, and found that both compounds are indistinguishable in their inhibition of anterograde transport and that both reagents significantly disrupt the morphology of the TGN in HeLa and in BS-C-1 cells. However, Exo2, unlike BFA, does not induce tubulation and merging of the TGN and endosomal compartments. Furthermore, and in contrast with its effects on cholera toxin, Exo2 significantly perturbs the delivery of Shiga toxin to the ER. Together, these results suggest that the likely target(s) of Exo2 operate at the level of the TGN, the Golgi and a subset of early endosomes, and thus Exo2 provides a more selective tool than BFA for examining membrane trafficking in mammalian cells.     

The essential role of PP1beta in Drosophila is to regulate nonmuscle myosin

Reversible phosphorylation of myosin regulatory light chain (MRLC) is a key regulatory mechanism controlling myosin activity and thus regulating the actin/myosin cytoskeleton. We show that Drosophila PP1beta, a specific isoform of serine/threonine protein phosphatase 1 (PP1), regulates nonmuscle myosin and that this is the essential role of PP1beta. Loss of PP1beta leads to increased levels of phosphorylated nonmuscle MRLC (Sqh) and actin disorganisation; these phenotypes can be suppressed by reducing the amount of active myosin. Drosophila has two nonmuscle myosin targeting subunits, one of which (MYPT-75D) resembles MYPT3, binds specifically to PP1beta, and activates PP1beta's Sqh phosphatase activity. Expression of a mutant form of MYPT-75D that is unable to bind PP1 results in elevation of Sqh phosphorylation in vivo and leads to phenotypes that can also be suppressed by reducing the amount of active myosin. The similarity between fly and human PP1beta and MYPT genes suggests this role may be conserved.     

Brefeldin A reversibly blocks early but not late protein transport steps in the yeast secretory pathway.

We have found that brefeldin A (BFA) inhibited the growth of an ise1 mutant of Saccharomyces cerevisiae. Genetic complementation and mapping studies demonstrated that ise1 was allelic to erg6, a gene required for the biosynthesis of the principal membrane sterol of yeast, ergosterol. Treatment of ise1 cells with BFA resulted in an immediate block in protein transport through the secretory pathway. Vacuolar carboxypeptidase Y (CPY) and the secreted pheromone alpha-factor accumulated as both the core glycosylated (ER) and alpha 1,6 mannosylated (early Golgi) forms in drug-treated cells. The modification of alpha-factor with alpha 1,6 mannose in BFA-treated cells did not appear to result from retrograde transport of the alpha 1,6 mannosyl-transferase into the ER. We found that transport of CPY from medial and late Golgi compartments to the vacuole was unaffected by BFA, nor was secretion of alpha 1,3 mannosylated alpha-factor or invertase blocked by BFA. The effects of BFA on the secretory pathway were also reversible after brief exposure (< 40 min) to the drug. We suggest that the primary effect of BFA in S. cerevisiae is restricted to the ER and the alpha 1,6 mannosyltransferase compartment of the Golgi complex.