Rho and Rac promote acinar morphological changes, actin reorganization, and amylase secretion

2005 ◽  
Vol 289 (3) ◽  
pp. G561-G570 ◽  
Author(s):  
Yan Bi ◽  
Sophie Le Page ◽  
John A. Williams
Keyword(s):  
Actin Polymerization ◽  
Morphological Changes ◽  
Dominant Negative ◽  
Amylase Secretion ◽  
Filamentous Actin ◽  
Pancreatic Acini ◽  
Actin Reorganization ◽  
Supramaximal Stimulation ◽  
Rho Family ◽  
High Concentrations

Supramaximal stimulation of isolated pancreatic acini with specific agonists such as CCK induces the formation of large basolateral blebs, redistributes filamentous actin, and inhibits secretion. Rho family small G proteins are well documented for their function in actin reorganization that determines cell shape and have been suggested to play a role in secretion. Here, we determined whether Rho and Rac are involved in the morphological changes, actin redistribution, and inhibition of amylase secretion induced by high concentrations of CCK. Introduction of constitutively active RhoV14 and RacV12 but not Cdc42V12 in mouse pancreatic acini by adenoviral vectors stimulated acinar morphological changes including basolateral protrusions, increased the total amount of F-actin, and reorganized the actin cytoskeleton. Dominant-negative RhoN19, Clostridium botulinum C3 exotoxin, which inhibits Rho, and dominant-negative RacN17 all partially blocked CCK-induced acinar morphological changes and actin redistribution. To study the correlation between actin polymerization and acinar shape changes, two marine toxins were employed. Jasplakinolide, a reagent that facilitates actin polymerization and stabilizes F-actin, stimulated acinar basolateral protrusions, whereas latrunculin, which sequesters actin monomers, blocked CCK-induced acinar blebbing. Unexpectedly, RhoV14, RacV12, and jasplakinolide all increased amylase secretion by CCK from 30 pM to 10 nM. The data suggest that Rho and Rac are involved in CCK-evoked changes in acinar morphology, actin redistribution, and secretion and that inhibition of secretion by high concentrations of CCK is not directly coupled to the changes in acinar morphology.

A role for Rho and Rac in secretagogue-induced amylase release by pancreatic acini

2005 ◽  
Vol 289 (1) ◽  
pp. C22-C32 ◽  
Author(s):  
Yan Bi ◽  
John A. Williams
Keyword(s):  
Actin Cytoskeleton ◽  
Late Phase ◽  
Inhibitory Effect ◽  
Pancreatic Acinar Cell ◽  
Dominant Negative ◽  
Amylase Secretion ◽  
Apical Region ◽  
Filamentous Actin ◽  
Amylase Release ◽  
Pancreatic Acini

The actin cytoskeleton has long been implicated in protein secretion. We investigated whether Rho and Rac, known regulators of the cytoskeleton, are involved in amylase secretion by mouse pancreatic acini. Secretagogues, including cholecystokinin (CCK) and the acetylcholine analog carbachol, increased the amount of GTP-bound RhoA and Rac1 and induced translocation from cytosol to a membrane fraction. Immunocytochemistry revealed the translocation of Rho and Rac within the apical region of the cell. Expression by means of adenoviral vectors of dominant-negative Rho (RhoN19), dominant-negative Rac (RacN17), and Clostridium Botulinum C3 exotoxin, which ADP ribosylates and inactivates Rho, significantly inhibited amylase secretion by CCK and carbachol; inhibiting both Rho and Rac resulted in a greater reduction. This inhibitory effect of RhoN19 on CCK-induced amylase secretion was apparent in both the early and late phases of secretion, whereas RacN17 was more potent on the late phase of secretion. None of these three affected the basal Ca2+ or the peak intracellular Ca2+ concentration stimulated by CCK. Latrunculin, a marine toxin that sequesters actin monomers, time-dependently decreased the total amount of filamentous actin (F-actin) and dose-dependently decreased secretion by secretagogues without affecting Ca2+ signaling. These data suggest that Rho and Rac are both involved in CCK-induced amylase release in pancreatic acinar cell possibly through an effect on the actin cytoskeleton.

scholarly journals Coxiella burnetii Induces Reorganization of the Actin Cytoskeleton in Human Monocytes

1998 ◽  
Vol 66 (11) ◽  
pp. 5527-5533 ◽  
Author(s):  
Sonia Meconi ◽  
Véronique Jacomo ◽  
Patrice Boquet ◽  
Didier Raoult ◽  
Jean-Louis Mege ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Coxiella Burnetii ◽  
Actin Polymerization ◽  
Q Fever ◽  
Morphological Changes ◽  
Critical Role ◽  
Filamentous Actin ◽  
Cytoskeleton Reorganization ◽  
Membrane Protrusions ◽  
Actin Protrusions

ABSTRACT Coxiella burnetii, an obligate intracellular bacterium which survives in myeloid cells, causes Q fever in humans. We previously demonstrated that virulent C. burnetiiorganisms are poorly internalized by monocytes compared to avirulent variants. We hypothesized that a differential mobilization of the actin cytoskeleton may account for this distinct phagocytic behavior. Scanning electron microscopy demonstrated that virulent C. burnetii stimulated profound and polymorphic changes in the morphology of THP-1 monocytes, consisting of membrane protrusions and polarized projections. These changes were transient, requiring 5 min to reach their maximum extent and vanishing after 60 min of incubation. In contrast, avirulent variants of C. burnetii did not induce any significant changes in cell morphology. The distribution of filamentous actin (F-actin) was then studied with a specific probe, bodipy phallacidin. Virulent C. burnetii induced a profound and transient reorganization of F-actin, accompanied by an increase in the F-actin content of THP-1 cells. F-actin was colocalized with myosin in cell protrusions, suggesting that actin polymerization and the tension of actin-myosin filaments play a role in C. burnetii-induced morphological changes. In addition, contact between the cell and the bacterium seems to be necessary to induce cytoskeleton reorganization. Bacterial supernatants did not stimulate actin remodeling, and virulent C. burnetii organisms were found in close apposition with F-actin protrusions. The manipulation of the actin cytoskeleton by C. burnetiimay therefore play a critical role in the internalization strategy of this bacterium.

scholarly journals A Cdc42 Activation Cycle Coordinated by PI 3-Kinase during Fc Receptor-mediated Phagocytosis

2010 ◽  
Vol 21 (3) ◽  
pp. 470-480 ◽  
Author(s):  
Peter Beemiller ◽  
Youxin Zhang ◽  
Suresh Mohan ◽  
Erik Levinsohn ◽  
Isabella Gaeta ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Plasma Membranes ◽  
Dominant Negative ◽  
Subsequent Increase ◽  
Pi3k Activation ◽  
Concentration Changes ◽  
Rho Family ◽  
Phagocytic Cups ◽  
Underlying Mechanisms ◽  
Activation Cycle

Fcγ Receptor (FcR)-mediated phagocytosis by macrophages requires phosphatidylinositol 3-kinase (PI3K) and activation of the Rho-family GTPases Cdc42 and Rac1. Cdc42 is activated at the advancing edge of the phagocytic cup, where actin is concentrated, and is deactivated at the base of the cup. The timing of 3′ phosphoinositide (3′PI) concentration changes in cup membranes suggests a role for 3′PIs in deactivation of Cdc42. This study examined the relationships between PI3K and the patterns of Rho-family GTPase signaling during phagosome formation. Inhibition of PI3K resulted in persistently active Cdc42 and Rac1, but not Rac2, in stalled phagocytic cups. Patterns of 3′PIs and Rho-family GTPase activities during phagocytosis of 5- and 2-μm-diameter microspheres indicated similar underlying mechanisms despite particle size–dependent sensitivities to PI3K inhibition. Expression of constitutively active Cdc42(G12V) increased 3′PI concentrations in plasma membranes and small phagosomes, indicating a role for Cdc42 in PI3K activation. Cdc42(G12V) inhibited phagocytosis at a later stage than inhibition by dominant negative Cdc42(N17). Together, these studies identified a Cdc42 activation cycle organized by PI3K, in which FcR-activated Cdc42 stimulates PI3K and actin polymerization, and the subsequent increase of 3′PIs in cup membranes inactivates Cdc42 to allow actin recycling necessary for phagosome formation.

Cholecystokinin-induced persistent stimulation of enzyme secretion from pancreatic acini

1981 ◽  
Vol 240 (6) ◽  
pp. G459-G465 ◽  
Author(s):  
S. M. Collins ◽  
S. Abdelmoumene ◽  
R. T. Jensen ◽  
J. D. Gardner
Keyword(s):  
Enzyme Secretion ◽  
Amylase Secretion ◽  
Intermediate Step ◽  
Pancreatic Acini ◽  
Persistent Effect ◽  
Incubation Solution ◽  
Cholecystokinin Receptors ◽  
Incomplete Removal ◽  
High Concentrations ◽  
Stimulation Of

When pancreatic acini are first incubated with cholecystokinin, washed to remove free cholecystokinin and then reincubated in fresh incubation solution, there is significant residual stimulation of amylase secretion. This residual stimulation requires relatively high concentrations of the secretagogue, is reversible, and is specific for cholecystokinin. Induction of residual stimulation occurs more rapidly at 37 degrees C (maximal by 1 min) than at 4 degrees C (maximal by 10 min), and, once induced, residual stimulation persists for up to 75 min at 37 degrees C and for more than 90 min at 4 degrees C. The persistent effect of cholecystokinin on enzyme secretion cannot be accounted for by incomplete removal of the secretagogue by the wash procedure or by activation of some intermediate step in the mechanism of action of cholecystokinin that persists after the secretagogue dissociates from its receptors. Instead, cholecystokinin-induced residual stimulation of enzyme secretion appears to result from persistent occupation of cholecystokinin receptors by the secretagogue.

scholarly journals Characterization of the megakaryocyte demarcation membrane system and its role in thrombopoiesis

Blood ◽  
2006 ◽  
Vol 107 (10) ◽  
pp. 3868-3875 ◽  
Author(s):  
Harald Schulze ◽  
Manav Korpal ◽  
Jonathan Hurov ◽  
Sang-We Kim ◽  
Jinghang Zhang ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Plasma Membranes ◽  
Blood Platelets ◽  
Membrane Surface ◽  
Cell Mass ◽  
Membrane System ◽  
Dominant Negative ◽  
Filamentous Actin ◽  
Local Assembly ◽  
Actin Expression

To produce blood platelets, megakaryocytes elaborate proplatelets, accompanied by expansion of membrane surface area and dramatic cytoskeletal rearrangements. The invaginated demarcation membrane system (DMS), a hallmark of mature cells, has been proposed as the source of proplatelet membranes. By direct visualization of labeled DMS, we demonstrate that this is indeed the case. Late in megakaryocyte ontogeny, the DMS gets loaded with PI-4,5-P2, a phospholipid that is confined to plasma membranes in other cells. Appearance of PI-4,5-P2 in the DMS occurs in proximity to PI-5-P-4-kinase α (PIP4Kα), and short hairpin (sh) RNA-mediated loss of PIP4Kα impairs both DMS development and expansion of megakaryocyte size. Thus, PI-4,5-P2 is a marker and possibly essential component of internal membranes. PI-4,5-P2 is known to promote actin polymerization by activating Rho-like GTPases and Wiskott-Aldrich syndrome (WASp) family proteins. Indeed, PI-4,5-P2 in the megakaryocyte DMS associates with filamentous actin. Expression of a dominant-negative N-WASp fragment or pharmacologic inhibition of actin polymerization causes similar arrests in proplatelet formation, acting at a step beyond expansion of the DMS and cell mass. These observations collectively suggest a signaling pathway wherein PI-4,5-P2 might facilitate DMS development and local assembly of actin fibers in preparation for platelet biogenesis.

scholarly journals The microtubule-binding protein CLIP-170 coordinates mDia1 and actin reorganization during CR3-mediated phagocytosis

2008 ◽  
Vol 183 (7) ◽  
pp. 1287-1298 ◽  
Author(s):  
Elodie Lewkowicz ◽  
Floriane Herit ◽  
Christophe Le Clainche ◽  
Pierre Bourdoncle ◽  
Franck Perez ◽  
...  
Keyword(s):  
Rna Interference ◽  
Actin Polymerization ◽  
Binding Protein ◽  
Receptor Activation ◽  
Microtubule Dynamics ◽  
Dominant Negative ◽  
Regulatory Proteins ◽  
Complement Receptor ◽  
Actin Reorganization

Microtubule dynamics are modulated by regulatory proteins that bind to their plus ends (+TIPs [plus end tracking proteins]), such as cytoplasmic linker protein 170 (CLIP-170) or end-binding protein 1 (EB1). We investigated the role of +TIPs during phagocytosis in macrophages. Using RNA interference and dominant-negative approaches, we show that CLIP-170 is specifically required for efficient phagocytosis triggered by αMβ2 integrin/complement receptor activation. This property is not observed for EB1 and EB3. Accordingly, whereas CLIP-170 is dynamically enriched at the site of phagocytosis, EB1 is not. Furthermore, we observe that CLIP-170 controls the recruitment of the formin mDia1, an actin-nucleating protein, at the onset of phagocytosis and thereby controls actin polymerization events that are essential for phagocytosis. CLIP-170 directly interacts with the formin homology 2 domain of mDia1. The interaction between CLIP-170 and mDia1 is negatively regulated during αMβ2-mediated phagocytosis. Our results unravel a new microtubule/actin cooperation that involves CLIP-170 and mDia1 and that functions downstream of αMβ2 integrins.

scholarly journals Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis

2011 ◽  
Vol 193 (5) ◽  
pp. 831-839 ◽  
Author(s):  
Michael Carnell ◽  
Tobias Zech ◽  
Simon D. Calaminus ◽  
Seiji Ura ◽  
Monica Hagedorn ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Dictyostelium Discoideum ◽  
Adenosine Triphosphatase ◽  
Actin Polymerization ◽  
Dominant Negative ◽  
Coated Vesicles ◽  
Filamentous Actin ◽  
Evolutionarily Conserved ◽  
Related Process

WASP and SCAR homologue (WASH) is a recently identified and evolutionarily conserved regulator of actin polymerization. In this paper, we show that WASH coats mature Dictyostelium discoideum lysosomes and is essential for exocytosis of indigestible material. A related process, the expulsion of the lethal endosomal pathogen Cryptococcus neoformans from mammalian macrophages, also uses WASH-coated vesicles, and cells expressing dominant negative WASH mutants inefficiently expel C. neoformans. D. discoideum WASH causes filamentous actin (F-actin) patches to form on lysosomes, leading to the removal of vacuolar adenosine triphosphatase (V-ATPase) and the neutralization of lysosomes to form postlysosomes. Without WASH, no patches or coats are formed, neutral postlysosomes are not seen, and indigestible material such as dextran is not exocytosed. Similar results occur when actin polymerization is blocked with latrunculin. V-ATPases are known to bind avidly to F-actin. Our data imply a new mechanism, actin-mediated sorting, in which WASH and the Arp2/3 complex polymerize actin on vesicles to drive the separation and recycling of proteins such as the V-ATPase.

scholarly journals Small GTPase RhoG Is a Key Regulator for Neurite Outgrowth in PC12 Cells

2000 ◽  
Vol 20 (19) ◽  
pp. 7378-7387 ◽  
Author(s):  
Hironori Katoh ◽  
Hidekazu Yasui ◽  
Yoshiaki Yamaguchi ◽  
Junko Aoki ◽  
Hirotada Fujita ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Morphological Changes ◽  
Cell Types ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Wild Type ◽  
Rho Family ◽  
Constitutively Active

ABSTRACT The Rho family of small GTPases has been implicated in cytoskeletal reorganization and subsequent morphological changes in various cell types. Among them, Rac and Cdc42 have been shown to be involved in neurite outgrowth in neuronal cells. In this study, we examined the role of RhoG, another member of Rho family GTPases, in nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Expression of wild-type RhoG in PC12 cells induced neurite outgrowth in the absence of NGF, and the morphology of wild-type RhoG-expressing cells was similar to that of NGF-differentiated cells. Constitutively active RhoG-transfected cells extended short neurites but developed large lamellipodial or filopodial structures at the tips of neurites. RhoG-induced neurite outgrowth was inhibited by coexpression with dominant-negative Rac1 or Cdc42. In addition, expression of constitutively active RhoG elevated endogenous Rac1 and Cdc42 activities. We also found that the NGF-induced neurite outgrowth was enhanced by expression of wild-type RhoG whereas expression of dominant-negative RhoG suppressed the neurite outgrowth. Furthermore, constitutively active Ras-induced neurite outgrowth was also suppressed by dominant-negative RhoG. Taken together, these results suggest that RhoG is a key regulator in NGF-induced neurite outgrowth, acting downstream of Ras and upstream of Rac1 and Cdc42 in PC12 cells.

Intracellular mediators of bombesin action on rat pancreatic acinar cells

1991 ◽  
Vol 260 (6) ◽  
pp. G858-G864 ◽  
Author(s):  
T. Matozaki ◽  
W. Y. Zhu ◽  
Y. Tsunoda ◽  
B. Goke ◽  
J. A. Williams
Keyword(s):  
Time Course ◽  
Pancreatic Acinar Cells ◽  
Amylase Secretion ◽  
Inhibitory Effects ◽  
Pancreatic Acini ◽  
Low Concentrations ◽  
Intracellular Messengers ◽  
Early Peak ◽  
High Concentrations ◽  
Intracellular Mediators

The effects of bombesin on physiological responses (amylase secretion, protein synthesis) and intracellular mediators [inositol 1,4,5-trisphosphate (1,4,5–IP3), [Ca2+]i, and diacylglycerol] were studied in isolated rat pancreatic acini and compared with the actions of cholecystokinin (CCK). Bombesin stimulated amylase secretion to the same extent as CCK. However, it failed to reproduce the inhibition of amylase secretion by high concentrations of CCK and likewise did not inhibit incorporation of [3H]leucine into protein in contrast to high concentrations of CCK. Low concentrations of bombesin (1–100 pM) induced repetitive oscillations in [Ca2+]i, whereas higher concentrations of bombesin (1–10 nM) induced a large transient increase in [Ca2+]i followed by a small sustained plateau. Bombesin (1–100 nM) induced an early peak of 1,4,5–IP3 at 5–15 s but was without measurable effect at lower concentrations. These effects on [Ca2+]i and 1,4,5–IP3 were similar to those seen with CCK except that bombesin was approximately 10-fold less potent than CCK. Bombesin induced an increase in acinar 1,2-diacylglycerol with a biphasic time course similar to CCK. However, the magnitude of the response to bombesin was much smaller than the response to CCK. The results suggest that bombesin receptors initiate similar intracellular messengers as does CCK. However, CCK induces a larger increase of diacylglycerol and probably an as yet unidentified messenger responsible for its inhibitory effects.

scholarly journals Function and interactions of the Ysc84/SH3yl1 family of actin- and lipid-binding proteins

2015 ◽  
Vol 43 (1) ◽  
pp. 111-116 ◽  
Author(s):  
Agnieszka N. Urbanek ◽  
Rebekah Chan ◽  
Kathryn R. Ayscough
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Regulatory Networks ◽  
Actin Polymerization ◽  
Binding Proteins ◽  
Morphological Changes ◽  
Lipid Binding ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Membrane Morphology

Understanding how actin filaments are nucleated, polymerized and disassembled in close proximity to cell membranes is an area of growing interest. Protrusion of the plasma membrane is required for cell motility, whereas inward curvature or invagination is required for endocytic events. These morphological changes in membrane are often associated with rearrangements of actin, but how the many actin-binding proteins of eukaryotes function in a co-ordinated way to generate the required responses is still not well understood. Identification and analysis of proteins that function at the interface between the plasma membrane and actin-regulatory networks is central to increasing our knowledge of the mechanisms required to transduce the force of actin polymerization to changes in membrane morphology. The Ysc84/SH3yl1 proteins have not been extensively studied, but work in both yeast and mammalian cells indicate that these proteins function at the hub of networks integrating regulation of filamentous actin (F-actin) with changes in membrane morphology.

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