scholarly journals Actin Filament Polymerization Regulates Gliding Motility by Apicomplexan Parasites

2003 ◽  
Vol 14 (2) ◽  
pp. 396-406 ◽  
Author(s):  
D.M. Wetzel ◽  
S. Håkansson ◽  
K. Hu ◽  
D. Roos ◽  
L.D. Sibley
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Gliding Motility ◽  
Cell Entry ◽  
Controlled Polymerization ◽  
Apicomplexan Parasites ◽  
Rate Limiting

Host cell entry by Toxoplasma gondii depends critically on actin filaments in the parasite, yet paradoxically, its actin is almost exclusively monomeric. In contrast to the absence of stable filaments in conventional samples, rapid-freeze electron microscopy revealed that actin filaments were formed beneath the plasma membrane of gliding parasites. To investigate the role of actin filaments in motility, we treated parasites with the filament-stabilizing drug jasplakinolide (JAS) and monitored the distribution of actin in live and fixed cells using yellow fluorescent protein (YFP)-actin. JAS treatment caused YFP-actin to redistribute to the apical and posterior ends, where filaments formed a spiral pattern subtending the plasma membrane. Although previous studies have suggested that JAS induces rigor, videomicroscopy demonstrated that JAS treatment increased the rate of parasite gliding by approximately threefold, indicating that filaments are rate limiting for motility. However, JAS also frequently reversed the normal direction of motility, disrupting forward migration and cell entry. Consistent with this alteration, subcortical filaments in JAS-treated parasites occurred in tangled plaques as opposed to the straight, roughly parallel orientation observed in control cells. These studies reveal that precisely controlled polymerization of actin filaments imparts the correct timing, duration, and directionality of gliding motility in the Apicomplexa.

Barrier role of actin filaments in regulated mucin secretion from airway goblet cells

2005 ◽  
Vol 288 (1) ◽  
pp. C46-C56 ◽  
Author(s):  
Camille Ehre ◽  
Andrea H. Rossi ◽  
Lubna H. Abdullah ◽  
Kathleen De Pestel ◽  
Sandra Hill ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Yellow Fluorescent Protein ◽  
Goblet Cells ◽  
Actin Binding ◽  
Secretory Cells ◽  
Cortical Actin ◽  
Mucin Secretion

Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/Gq-coupled P2Y2receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of β- and γ-actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of β- or γ-actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca2+-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis.

scholarly journals GLP-1 Receptor Stimulation of the Lateral Parabrachial Nucleus Reduces Food Intake: Neuroanatomical, Electrophysiological, and Behavioral Evidence

Endocrinology ◽  
2014 ◽  
Vol 155 (11) ◽  
pp. 4356-4367 ◽  
Author(s):  
Jennifer E. Richard ◽  
Imre Farkas ◽  
Fredrik Anesten ◽  
Rozita H. Anderberg ◽  
Suzanne L. Dickson ◽  
...  
Keyword(s):  
Feeding Behavior ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Parabrachial Nucleus ◽  
Lateral Parabrachial Nucleus ◽  
Electrophysiological Studies ◽  
Glucagon Like Peptide 1 ◽  
Behavioral Evidence ◽  
Stimulation Of

Abstract The parabrachial nucleus (PBN) is a key nucleus for the regulation of feeding behavior. Inhibitory inputs from the hypothalamus to the PBN play a crucial role in the normal maintenance of feeding behavior, because their loss leads to starvation. Viscerosensory stimuli result in neuronal activation of the PBN. However, the origin and neurochemical identity of the excitatory neuronal input to the PBN remain largely unexplored. Here, we hypothesize that hindbrain glucagon-like peptide 1 (GLP-1) neurons provide excitatory inputs to the PBN, activation of which may lead to a reduction in feeding behavior. Our data, obtained from mice expressing the yellow fluorescent protein in GLP-1-producing neurons, revealed that hindbrain GLP-1-producing neurons project to the lateral PBN (lPBN). Stimulation of lPBN GLP-1 receptors (GLP-1Rs) reduced the intake of chow and palatable food and decreased body weight in rats. It also activated lPBN neurons, reflected by an increase in the number of c-Fos-positive cells in this region. Further support for an excitatory role of GLP-1 in the PBN is provided by electrophysiological studies showing a remarkable increase in firing of lPBN neurons after Exendin-4 application. We show that within the PBN, GLP-1R activation increased gene expression of 2 energy balance regulating peptides, calcitonin gene-related peptide (CGRP) and IL-6. Moreover, nearly 70% of the lPBN GLP-1 fibers innervated lPBN CGRP neurons. Direct intra-lPBN CGRP application resulted in anorexia. Collectively, our molecular, anatomical, electrophysiological, pharmacological, and behavioral data provide evidence for a functional role of the GLP-1R for feeding control in the PBN.

scholarly journals Anchoring of actin to the plasma membrane enables tension production in the fission yeast cytokinetic ring

2019 ◽  
Vol 30 (16) ◽  
pp. 2053-2064 ◽  
Author(s):  
Shuyuan Wang ◽  
Ben O’Shaughnessy
Keyword(s):  
Plasma Membrane ◽  
Fission Yeast ◽  
Actin Filaments ◽  
Tensile Force ◽  
Myosin Ii ◽  
Quantitative Evidence ◽  
Explicit Simulation ◽  
Adjacent Segments ◽  
Develop Tension

The cytokinetic ring generates tensile force that drives cell division, but how tension emerges from the relatively disordered ring organization remains unclear. Long ago, a musclelike sliding filament mechanism was proposed, but evidence for sarcomeric order is lacking. Here we present quantitative evidence that in fission yeast, ring tension originates from barbed-end anchoring of actin filaments to the plasma membrane, providing resistance to myosin forces that enables filaments to develop tension. The role of anchoring was highlighted by experiments on isolated fission yeast rings, where sections of ring became unanchored from the membrane and shortened ∼30-fold faster than normal. The dramatically elevated constriction rates are unexplained. Here we present a molecularly explicit simulation of constricting partially anchored rings as studied in these experiments. Simulations accurately reproduced the experimental constriction rates and showed that following anchor release, a segment becomes tensionless and shortens via a novel noncontractile reeling-in mechanism at about the velocity of load-free myosin II. The ends are reeled in by barbed end–anchored actin filaments in adjacent segments. Other actin anchoring schemes failed to constrict rings. Our results quantitatively support a specific organization and anchoring scheme that generate tension in the cytokinetic ring.

scholarly journals Investigating the Role of F-Actin in Human Immunodeficiency Virus Assembly by Live-Cell Microscopy

2014 ◽  
Vol 88 (14) ◽  
pp. 7904-7914 ◽  
Author(s):  
Sheikh Abdul Rahman ◽  
Peter Koch ◽  
Julian Weichsel ◽  
William J. Godinez ◽  
Ulrich Schwarz ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Virus Assembly ◽  
Live Cell ◽  
Viral Budding ◽  
Immunodeficiency Virus ◽  
Live Cell Microscopy ◽  
Gag Assembly ◽  
Hiv 1

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) particles assemble at the plasma membrane, which is lined by a dense network of filamentous actin (F-actin). Large amounts of actin have been detected in HIV-1 virions, proposed to be incorporated by interactions with the nucleocapsid domain of the viral polyprotein Gag. Previous studies addressing the role of F-actin in HIV-1 particle formation using F-actin-interfering drugs did not yield consistent results. Filamentous structures pointing toward nascent HIV-1 budding sites, detected by cryo-electron tomography and atomic force microscopy, prompted us to revisit the role of F-actin in HIV-1 assembly by live-cell microscopy. HeLa cells coexpressing HIV-1 carrying fluorescently labeled Gag and a labeled F-actin-binding peptide were imaged by live-cell total internal reflection fluorescence microscopy (TIR-FM). Computational analysis of image series did not reveal characteristic patterns of F-actin in the vicinity of viral budding sites. Furthermore, no transient recruitment of F-actin during bud formation was detected by monitoring fluorescence intensity changes at nascent HIV-1 assembly sites. The chosen approach allowed us to measure the effect of F-actin-interfering drugs on the assembly of individual virions in parallel with monitoring changes in the F-actin network of the respective cell. Treatment of cells with latrunculin did not affect the efficiency and dynamics of Gag assembly under conditions resulting in the disruption of F-actin filaments. Normal assembly rates were also observed upon transient stabilization of F-actin by short-term treatment with jasplakinolide. Taken together, these findings indicate that actin filament dynamics are dispensable for HIV-1 Gag assembly at the plasma membrane of HeLa cells.IMPORTANCEHIV-1 particles assemble at the plasma membrane of virus-producing cells. This membrane is lined by a dense network of actin filaments that might either present a physical obstacle to the formation of virus particles or generate force promoting the assembly process. Drug-mediated interference with the actin cytoskeleton showed different results for the formation of retroviral particles in different studies, likely due to general effects on the cell upon prolonged drug treatment. Here, we characterized the effect of actin-interfering compounds on the HIV-1 assembly process by direct observation of virus formation in live cells, which allowed us to measure assembly rate constants directly upon drug addition. Virus assembly proceeded with normal rates when actin filaments were either disrupted or stabilized. Taken together with the absence of characteristic actin filament patterns at viral budding sites in our analyses, this indicates that the actin network is dispensable for HIV-1 assembly.

Plasma membrane delivery of the gastric H,K-ATPase: the role of β-subunit glycosylation

2003 ◽  
Vol 285 (4) ◽  
pp. C968-C976 ◽  
Author(s):  
O. Vagin ◽  
S. Denevich ◽  
G. Sachs
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Glycosylation Site ◽  
Β Subunit ◽  
Wild Type ◽  
Α Subunit ◽  
Hek 293 ◽  
Glycosylation Sites ◽  
293 Cells

The factors determining trafficking of the gastric H,K-ATPase to the apical membrane remain elusive. To identify such determinants in the gastric H,K-ATPase, fusion proteins of yellow fluorescent protein (YFP) and the gastric H,K-ATPase β-subunit (YFP-β) and cyan fluorescent protein (CFP) and the gastric H,K-ATPase α-subunit (CFP-α) were expressed in HEK-293 cells. Then plasma membrane delivery of wild-type CFP-α, wild-type YFP-β, and YFP-β mutants lacking one or two of the seven β-subunit glycosylation sites was determined using confocal microscopy and surface biotinylation. Expression of the wild-type YFP-β resulted in the plasma membrane localization of the protein, whereas the expressed CFP-α was retained intracellularly. When coexpressed, both CFP-α and YFP-β were delivered to the plasma membrane. Removing each of the seven glycosylation sites, except the second one, from the extracellular loop of YFP-β prevented plasma membrane delivery of the protein. Only the mutant lacking the second glycosylation site (Asn103Gln) was localized both intracellularly and on the plasma membrane. A double mutant lacking the first (Asn99Gln) and the second (Asn103Gln) glycosylation sites displayed intracellular accumulation of the protein. Therefore, six of the seven glycosylation sites in the β-subunit are essential for the plasma membrane delivery of the β-subunit of the gastric H,K-ATPase, whereas the second glycosylation site (Asn103), which is not conserved among the β-subunits from different species, is not critical for plasma delivery of the protein.

scholarly journals The Multiple Roles of Cyk1p in the Assembly and Function of the Actomyosin Ring in Budding Yeast

1999 ◽  
Vol 10 (2) ◽  
pp. 283-296 ◽  
Author(s):  
Katie B. Shannon ◽  
Rong Li
Keyword(s):  
Actin Filaments ◽  
Fluorescent Protein ◽  
Budding Yeast ◽  
Dependent Manner ◽  
Size Change ◽  
Direct Role ◽  
Dynamic Component ◽  
Amino Terminal ◽  
Ring Assembly

The budding yeast IQGAP-like protein Cyk1p/Iqg1p localizes to the mother-bud junction during anaphase and has been shown to be required for the completion of cytokinesis. In this study, video microscopy analysis of cells expressing green fluorescent protein-tagged Cyk1p/Iqg1p demonstrates that Cyk1p/Iqg1p is a dynamic component of the contractile ring during cytokinesis. Furthermore, in the absence of Cyk1p/Iqg1p, myosin II fails to undergo the contraction-like size change at the end of mitosis. To understand the mechanistic role of Cyk1p/Iqg1p in actomyosin ring assembly and dynamics, we have investigated the role of the structural domains that Cyk1p/Iqg1p shares with IQGAPs. An amino terminal portion containing the calponin homology domain binds to actin filaments and is required for the assembly of actin filaments to the ring. This result supports the hypothesis that Cyk1p/Iqg1p plays a direct role in F-actin recruitment. Deletion of the domain harboring the eight IQ motifs abolishes the localization of Cyk1p/Iqg1p to the bud neck, suggesting that Cyk1p/Iqg1p may be localized through interactions with a calmodulin-like protein. Interestingly, deletion of the COOH-terminal GTPase-activating protein-related domain does not affect Cyk1p/Iqg1p localization or actin recruitment to the ring but prevents actomyosin ring contraction. In vitro binding experiments show that Cyk1p/Iqg1p binds to calmodulin, Cmd1p, in a calcium-dependent manner, and to Tem1p, a small GTP-binding protein previously found to be required for the completion of anaphase. These results demonstrate the critical function of Cyk1p/Iqg1p in regulating various steps of actomyosin ring assembly and cytokinesis.

scholarly journals Construction and Characterization of a Lactose-Inducible Promoter System for Controlled Gene Expression inClostridium perfringens

2010 ◽  
Vol 77 (2) ◽  
pp. 471-478 ◽  
Author(s):  
Andrea H. Hartman ◽  
Hualan Liu ◽  
Stephen B. Melville
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Shuttle Vector ◽  
Yellow Fluorescent Protein ◽  
Inducible Promoter ◽  
Gliding Motility ◽  
Glucuronidase Activity ◽  
Atpase Gene ◽  
Regulated Gene Expression ◽  
Promoter System

ABSTRACTClostridium perfringensis a Gram-positive anaerobic pathogen which causes many diseases in humans and animals. While some genetic tools exist for working withC. perfringens, a tightly regulated, inducible promoter system is currently lacking. Therefore, we constructed a plasmid-based promoter system that provided regulated expression when lactose was added. This plasmid (pKRAH1) is anEscherichia coli-C. perfringensshuttle vector containing the gene encoding a transcriptional regulator, BgaR, and a divergent promoter upstream of genebgaL(bgaR-PbgaL). To measure transcription at thebgaLpromoter in pKRAH1, theE. colireporter genegusA, encoding β-glucuronidase, was placed downstream of the PbgaLpromoter to make plasmid pAH2. When transformed into three strains ofC. perfringens, pAH2 exhibited lactose-inducible expression.C. perfringensstrain 13, a commonly studied strain, has endogenous β-glucuronidase activity. We mutated genebglR, encoding a putative β-glucuronidase, and observed an 89% decrease in endogenous activity with no lactose. This combination of a system for regulated gene expression and a mutant of strain 13 with low β-glucuronidase activity are useful tools for studying gene regulation and protein expression in an important pathogenic bacterium. We used this system to express theyfp-pilBgene, comprised of a yellow fluorescent protein (YFP)-encoding gene fused to an assembly ATPase gene involved in type IV pilus-dependent gliding motility inC. perfringens. Expression in the wild-type strain showed that YFP-PilB localized mostly to the poles of cells, but in apilCmutant it localized throughout the cell, demonstrating that the membrane protein PilC is required for polar localization of PilB.

scholarly journals Conventional Kinesin Mediates Microtubule-Microtubule Interactions In Vivo

2006 ◽  
Vol 17 (2) ◽  
pp. 907-916 ◽  
Author(s):  
Anne Straube ◽  
Gerd Hause ◽  
Gero Fink ◽  
Gero Steinberg
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Binding Activity ◽  
C Terminus ◽  
Cross Bridges ◽  
Conventional Kinesin ◽  
Motor Head

Conventional kinesin is a ubiquitous organelle transporter that moves cargo toward the plus-ends of microtubules. In addition, several in vitro studies indicated a role of conventional kinesin in cross-bridging and sliding microtubules, but in vivo evidence for such a role is missing. In this study, we show that conventional kinesin mediates microtubule-microtubule interactions in the model fungus Ustilago maydis. Live cell imaging and ultrastructural analysis of various mutants in Kin1 revealed that this kinesin-1 motor is required for efficient microtubule bundling and participates in microtubule bending in vivo. High levels of Kin1 led to increased microtubule bending, whereas a rigor-mutation in the motor head suppressed all microtubule motility and promoted strong microtubule bundling, indicating that kinesin can form cross-bridges between microtubules in living cells. This effect required a conserved region in the C terminus of Kin1, which was shown to bind microtubules in vitro. In addition, a fusion protein of yellow fluorescent protein and the Kin1tail localized to microtubule bundles, further supporting the idea that a conserved microtubule binding activity in the tail of conventional kinesins mediates microtubule-microtubule interactions in vivo.

scholarly journals Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii

2011 ◽  
Vol 22 (8) ◽  
pp. 1290-1299 ◽  
Author(s):  
Simren Mehta ◽  
L. David Sibley
Keyword(s):  
Toxoplasma Gondii ◽  
Actin Filaments ◽  
Gliding Motility ◽  
Conditional Knockout ◽  
Host Cells ◽  
Actin Binding ◽  
Actin Depolymerizing Factor

Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ∼98% of actin is nonfilamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role of actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii actin depolymerizing factor (TgADF) has strong actin monomer–sequestering and weak filament-severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and electron microscopy. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion.

scholarly journals Role of phosphoinositides in STIM1 dynamics and store-operated calcium entry

2009 ◽  
Vol 425 (1) ◽  
pp. 159-168 ◽  
Author(s):  
Ciara M. Walsh ◽  
Michael Chvanov ◽  
Lee P. Haynes ◽  
Ole H. Petersen ◽  
Alexei V. Tepikin ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Phosphatidylinositol 3 Kinase ◽  
Enhanced Yellow Fluorescent Protein ◽  
Store Depletion ◽  
Store Operated Calcium Entry ◽  
Translocation Mechanism ◽  
Direct Binding ◽  
Phosphatidylinositol 4

Ca2+ entry through store-operated Ca2+ channels involves the interaction at ER–PM (endoplasmic reticulum–plasma membrane) junctions of STIM (stromal interaction molecule) and Orai. STIM proteins are sensors of the luminal ER Ca2+ concentration and, following depletion of ER Ca2+, they oligomerize and translocate to ER–PM junctions where they form STIM puncta. Direct binding to Orai proteins activates their Ca2+ channel function. It has been suggested that an additional interaction of the C-terminal polybasic domain of STIM1 with PM phosphoinositides could contribute to STIM1 puncta formation prior to binding to Orai. In the present study, we investigated the role of phosphoinositides in the formation of STIM1 puncta and SOCE (store-operated Ca2+ entry) in response to store depletion. Treatment of HeLa cells with inhibitors of PI3K (phosphatidylinositol 3-kinase) and PI4K (phosphatidylinositol 4-kinase) (wortmannin and LY294002) partially inhibited formation of STIM1 puncta. Additional rapid depletion of PtdIns(4,5)P2 resulted in more substantial inhibition of the translocation of STIM1–EYFP (enhanced yellow fluorescent protein) into puncta. The inhibition was extensive at a concentration of LY294002 (50 μM) that should primarily inhibit PI3K, consistent with a major role for PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in puncta formation. Depletion of phosphoinositides also inhibited SOCE based on measurement of the rise in intracellular Ca2+ concentration after store depletion. Overexpression of Orai1 resulted in a recovery of translocation of STMI1 into puncta following phosphoinositide depletion and, under these conditions, SOCE was increased to above control levels. These observations support the idea that phosphoinositides are not essential but contribute to STIM1 accumulation at ER–PM junctions with a second translocation mechanism involving direct STIM1–Orai interactions.

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