scholarly journals Formation and disassembly of a contractile actomyosin network mediates content release from large secretory vesicles

2017 ◽  
Author(s):  
Dagan Segal ◽  
Assaf Zaritsky ◽  
Eyal D. Schejter ◽  
Ben-Zion Shilo
Keyword(s):  
Salivary Glands ◽  
Negative Feedback ◽  
Secretory Vesicles ◽  
Actin Assembly ◽  
Actin Nucleation ◽  
Actin Turnover ◽  
Apical Membranes ◽  
Contraction Cycle ◽  
Actomyosin Contraction

AbstractSecretion of adhesive glycoproteins to the lumen of Drosophila larval salivary glands is carried out by contraction of an actomyosin network that is assembled around large secretory vesicles, following their fusion to the apical membranes. We have identified a cycle of actin coat nucleation and disassembly that is independent of myosin. Recruitment of active Rho1 to the fused vesicle triggers activation of the formin Diaphanous and nucleation of linear actin. This, in turn, leads to actin-dependent localization of a RhoGAP protein that locally shuts off Rho1, promoting disassembly of the actin coat. Recruitment of the branched actin nucleation machinery is also required for effective Rho1 inactivation. Interestingly, different blocks to actin coat disassembly arrested vesicle contraction, indicating that actin turnover is an integral part of the actomyosin contraction cycle. The capacity of F-actin to trigger a negative feedback on its own production may be utilized in a variety of scenarios, to coordinate a succession of morphogenetic events or maintain homeostasis.SummaryThis work identified a cycle of actin assembly and disassembly in large secretory vesicles of Drosophila salivary glands. Actin disassembly is triggered by actin-dependent recruitment of a RhoGAP protein, and is essential for the contractility of the vesicle leading to content release to the lumen.

scholarly journals Feedback inhibition of actin on Rho mediates content release from large secretory vesicles

2018 ◽  
Vol 217 (5) ◽  
pp. 1815-1826 ◽  
Author(s):  
Dagan Segal ◽  
Assaf Zaritsky ◽  
Eyal D. Schejter ◽  
Ben-Zion Shilo
Keyword(s):  
Drosophila Melanogaster ◽  
Salivary Glands ◽  
Negative Feedback ◽  
Temporal Order ◽  
Feedback Inhibition ◽  
Secretory Vesicles ◽  
Actin Turnover ◽  
Apical Membranes ◽  
Contraction Cycle ◽  
Actomyosin Contraction

Secretion of adhesive glycoproteins to the lumen of Drosophila melanogaster larval salivary glands is performed by contraction of an actomyosin network assembled around large secretory vesicles, after their fusion to the apical membranes. We have identified a cycle of actin coat nucleation and disassembly that is independent of myosin. Recruitment of active Rho1 to the fused vesicle triggers activation of the formin Diaphanous and actin nucleation. This leads to actin-dependent localization of a RhoGAP protein that locally shuts off Rho1, promoting disassembly of the actin coat. When contraction of vesicles is blocked, the strict temporal order of the recruited elements generates repeated oscillations of actin coat formation and disassembly. Interestingly, different blocks to actin coat disassembly arrested vesicle contraction, indicating that actin turnover is an integral part of the actomyosin contraction cycle. The capacity of F-actin to trigger a negative feedback on its own production may be widely used to coordinate a succession of morphogenetic events or maintain homeostasis.

Ultrastructural Analysis of the Salivary Glands of Gromphadorhina Portentosa (Schaum)

1977 ◽  
Vol 35 ◽  
pp. 638-639
Author(s):  
P.J. Dailey
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Salivary Glands ◽  
Secretory Vesicles ◽  
The Past ◽  
Transmission Electron ◽  
Means Of Transmission ◽  
Gromphadorhina Portentosa ◽  
Scanning Electron ◽  
Topographical Relief

The structure of insect salivary glands has been extensively investigated during the past decade; however, none have attempted scanning electron microscopy (SEM) in ultrastructural examinations of these secretory organs. This study correlates fine structure by means of SEM cryofractography with that of thin-sectioned epoxy embedded material observed by means of transmission electron microscopy (TEM).Salivary glands of Gromphadorhina portentosa were excised and immediately submerged in cold (4°C) paraformaldehyde-glutaraldehyde fixative1 for 2 hr, washed and post-fixed in 1 per cent 0s04 in phosphosphate buffer (4°C for 2 hr). After ethanolic dehydration half of the samples were embedded in Epon 812 for TEM and half cryofractured and subsequently critical point dried for SEM. Dried specimens were mounted on aluminum stubs and coated with approximately 150 Å of gold in a cold sputtering apparatus.Figure 1 shows a cryofractured plane through a salivary acinus revealing topographical relief of secretory vesicles.

scholarly journals Immunolocalization of electroneutral Na+-HCO 3 − cotransporters in human and rat salivary glands

2002 ◽  
Vol 283 (2) ◽  
pp. G473-G480 ◽  
Author(s):  
V. Gresz ◽  
T.-H. Kwon ◽  
H. Vorum ◽  
T. Zelles ◽  
I. Kurtz ◽  
...  
Keyword(s):  
Salivary Glands ◽  
Basolateral Membrane ◽  
Salivary Secretion ◽  
Rat Tissues ◽  
Submandibular Glands ◽  
Rat Salivary Glands ◽  
Duct Cells ◽  
Mrna And Protein Expression ◽  
Rat Parotid ◽  
Apical Membranes

Patterns of salivary HCO[Formula: see text]secretion vary widely among species and among individual glands. In particular, virtually nothing is known about the molecular identity of the HCO[Formula: see text] transporters involved in human salivary secretion. We have therefore examined the distribution of several known members of the Na+-HCO[Formula: see text] cotransporter (NBC) family in the parotid and submandibular glands. By use of a combination of RT-PCR and immunoblotting analyses, the electroneutral cotransporters NBC3 and NBCn1 mRNA and protein expression were detected in both human and rat tissues. Immunohistochemistry demonstrated that NBC3 was present at the apical membranes of acinar and duct cells in both human and rat parotid and submandibular glands. NBCn1 was strongly expressed at the basolateral membrane of striated duct cells but not in the acinar cells in the human salivary glands, whereas little or no NBCn1 labeling was observed in the rat salivary glands. The presence of NBCn1 at the basolateral membrane of human striated duct cells suggests that it may contribute to ductal HCO[Formula: see text] secretion. In contrast, the expression of NBC3 at the apical membranes of acinar and duct cells in both human and rat salivary glands indicates a possible role of this isoform in HCO[Formula: see text] salvage under resting conditions.

scholarly journals WAVE1 and WAVE2 have distinct and overlapping roles in controlling actin assembly at the leading edge

2020 ◽  
Vol 31 (20) ◽  
pp. 2168-2178
Author(s):  
Qing Tang ◽  
Matthias Schaks ◽  
Neha Koundinya ◽  
Changsong Yang ◽  
Luther W. Pollard ◽  
...  
Keyword(s):  
Leading Edge ◽  
Actin Assembly ◽  
Actin Network ◽  
Actin Nucleation ◽  
Lamellipodia Formation ◽  
Network Extension

This study shows that WAVE1 and WAVE2 have redundant roles as actin nucleation-promoting factors (NPFs) in promoting lamellipodia formation, but also unique and nonoverlapping roles in controlling the rate of actin network extension, with WAVE2 promoting and WAVE1 restricting growth.

scholarly journals Rab1 recruits WHAMM during membrane remodeling but limits actin nucleation

2016 ◽  
Vol 27 (6) ◽  
pp. 967-978 ◽  
Author(s):  
Ashley J. Russo ◽  
Alyssa J. Mathiowetz ◽  
Steven Hong ◽  
Matthew D. Welch ◽  
Kenneth G. Campellone
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Actin Assembly ◽  
Membrane Remodeling ◽  
Actin Nucleation ◽  
Factor Interactions ◽  
Cytoskeletal Rearrangements ◽  
Distinct Strategy ◽  
Small G Proteins

Small G-proteins are key regulatory molecules that activate the actin nucleation machinery to drive cytoskeletal rearrangements during plasma membrane remodeling. However, the ability of small G-proteins to interact with nucleation factors on internal membranes to control trafficking processes has not been well characterized. Here we investigated roles for members of the Rho, Arf, and Rab G-protein families in regulating WASP homologue associated with actin, membranes, and microtubules (WHAMM), an activator of Arp2/3 complex–mediated actin nucleation. We found that Rab1 stimulated the formation and elongation of WHAMM-associated membrane tubules in cells. Active Rab1 recruited WHAMM to dynamic tubulovesicular structures in fibroblasts, and an active prenylated version of Rab1 bound directly to an N-terminal domain of WHAMM in vitro. In contrast to other G-protein–nucleation factor interactions, Rab1 binding inhibited WHAMM-mediated actin assembly. This ability of Rab1 to regulate WHAMM and the Arp2/3 complex represents a distinct strategy for membrane remodeling in which a Rab G-protein recruits the actin nucleation machinery but dampens its activity.

scholarly journals Kiss-and-Coat and Compartment Mixing: Coupling Exocytosis to Signal Generation and Local Actin Assembly

2006 ◽  
Vol 17 (4) ◽  
pp. 1495-1502 ◽  
Author(s):  
Anna M. Sokac ◽  
William M. Bement
Keyword(s):  
Cell Types ◽  
Secretory Vesicle ◽  
Signal Generation ◽  
General Mechanism ◽  
Fusion Pore ◽  
Secretory Vesicles ◽  
Actin Assembly ◽  
Vesicle Membrane ◽  
Regulated Exocytosis ◽  
Broad Variety

Regulated exocytosis is thought to occur either by “full fusion,” where the secretory vesicle fuses with the plasma membrane (PM) via a fusion pore that then dilates until the secretory vesicle collapses into the PM; or by “kiss-and-run,” where the fusion pore does not dilate and instead rapidly reseals such that the secretory vesicle is retrieved almost fully intact. Here, we describe growing evidence for a third form of exocytosis, dubbed “kiss-and-coat,” which is characteristic of a broad variety of cell types that undergo regulated exocytosis. Kiss-and-coat exocytosis entails prolonged maintenance of a dilated fusion pore and assembly of actin filament (F-actin) coats around the exocytosing secretory vesicles followed by direct retrieval of some fraction of the emptied vesicle membrane. We propose that assembly of the actin coats results from the union of the secretory vesicle membrane and PM and that this compartment mixing represents a general mechanism for generating local signals via directed membrane fusion.

scholarly journals Cell migration without a lamellipodium

2005 ◽  
Vol 168 (4) ◽  
pp. 619-631 ◽  
Author(s):  
Stephanie L. Gupton ◽  
Karen L. Anderson ◽  
Thomas P. Kole ◽  
Robert S. Fischer ◽  
Aaron Ponti ◽  
...  
Keyword(s):  
Cell Migration ◽  
Epithelial Cells ◽  
Leading Edge ◽  
Actin Binding ◽  
Retrograde Flow ◽  
Actin Assembly ◽  
Migration Inhibition ◽  
Actin Networks ◽  
Actin Turnover ◽  
High Concentrations

The actin cytoskeleton is locally regulated for functional specializations for cell motility. Using quantitative fluorescent speckle microscopy (qFSM) of migrating epithelial cells, we previously defined two distinct F-actin networks based on their F-actin–binding proteins and distinct patterns of F-actin turnover and movement. The lamellipodium consists of a treadmilling F-actin array with rapid polymerization-dependent retrograde flow and contains high concentrations of Arp2/3 and ADF/cofilin, whereas the lamella exhibits spatially random punctae of F-actin assembly and disassembly with slow myosin-mediated retrograde flow and contains myosin II and tropomyosin (TM). In this paper, we microinjected skeletal muscle αTM into epithelial cells, and using qFSM, electron microscopy, and immunolocalization show that this inhibits functional lamellipodium formation. Cells with inhibited lamellipodia exhibit persistent leading edge protrusion and rapid cell migration. Inhibition of endogenous long TM isoforms alters protrusion persistence. Thus, cells can migrate with inhibited lamellipodia, and we suggest that TM is a major regulator of F-actin functional specialization in migrating cells.

scholarly journals Dictyostelium discoideum plasma membranes contain an actin-nucleating activity that requires ponticulin, an integral membrane glycoprotein.

1990 ◽  
Vol 110 (3) ◽  
pp. 681-692 ◽  
Author(s):  
A Shariff ◽  
E J Luna
Keyword(s):  
Dictyostelium Discoideum ◽  
Actin Polymerization ◽  
Plasma Membranes ◽  
Actin Binding ◽  
Heat Denaturation ◽  
Actin Assembly ◽  
Binding Studies ◽  
Actin Nucleation ◽  
Equilibrium Binding ◽  
Nucleation Activity

In previous equilibrium binding studies, Dictyostelium discoideum plasma membranes have been shown to bind actin and to recruit actin into filaments at the membrane surface. However, little is known about the kinetic pathway(s) through which actin assembles at these, or other, membranes. We have used actin fluorescently labeled with N-(1-pyrenyl)iodoacetamide to examine the kinetics of actin assembly in the presence of D. discoideum plasma membranes. We find that these membranes increase the rate of actin polymerization. The rate of membrane-mediated actin polymerization is linearly dependent on membrane protein concentrations up to 20 micrograms/ml. Nucleation (the association of activated actin monomers into oligomers) appears to be the primary step of polymerization that is accelerated. A sole effect on the initial salt-induced actin conformational change (activation) is ruled out because membranes accelerate the polymerization of pre-activated actin as well as actin activated in the presence of membranes. Elongation of preexisting filaments also is not the major step of polymerization facilitated by membranes since membranes stripped of all peripheral components, including actin, increase the rate of actin assembly to about the same extent as do membranes containing small amounts of endogenous actin. Acceleration of the nucleation step by membranes also is supported by an analysis of the dependence of polymerization lag time on actin concentration. The barbed ends of membrane-induced actin nuclei are not obstructed by the membranes because the barbed end blocking agent, cytochalasin D, reduces the rate of membrane-mediated actin nucleation. Similarly, the pointed ends of the nuclei are not blocked by membranes since the depolymerization rate of gelsolin-capped actin is unchanged in the presence of membranes. These results are consistent with previous observations of lateral interactions between membranes and actin filaments. These results also are consistent with two predictions from a model based on equilibrium binding studies; i.e., that plasma membranes should nucleate actin assembly and that membrane-bound actin nuclei should have both ends free (Schwartz, M. A., and E. J. Luna. 1988. J. Cell Biol. 107:201-209). Integral membrane proteins mediate the actin nucleation activity because activity is eliminated by heat denaturation, treatment with reducing agents, or proteolysis of membranes. Activity also is abolished by solubilization with octylglucoside but is reconstituted upon removal or dilution of the detergent. Ponticulin, the major actin-binding protein in plasma membranes, appears to be necessary for nucleation activity since activity is not reconstituted from detergent extracts depleted of ponticulin.

scholarly journals MICAL2 acts through Arp3B isoform-specific Arp2/3 complexes to destabilize branched actin networks

2020 ◽  
Author(s):  
Chiara Galloni ◽  
Davide Carra ◽  
Jasmine V. G. Abella ◽  
Svend Kjær ◽  
Pavithra Singaravelu ◽  
...  
Keyword(s):  
Functional Properties ◽  
Actin Filament ◽  
Actin Assembly ◽  
Actin Network ◽  
Network Stability ◽  
Actin Networks ◽  
Cellular Processes ◽  
Actin Turnover ◽  
Filament Networks

AbstractThe Arp2/3 complex (Arp2, Arp3 and ARPC1-5) is essential to generate branched actin filament networks for many cellular processes. Human Arp3, ARPC1 and ARPC5 exist as two isoforms but the functional properties of Arp2/3 iso-complexes is largely unexplored. Here we show that Arp3B, but not Arp3 is subject to regulation by the methionine monooxygenase MICAL2, which is recruited to branched actin networks by coronin-1C. Although Arp3 and Arp3B iso-complexes promote actin assembly equally efficiently in vitro, they have different cellular properties. Arp3B turns over significantly faster than Arp3 within the network and upon its depletion actin turnover decreases. Substitution of Arp3B Met293 by Thr, the corresponding residue in Arp3 increases actin network stability, and conversely, replacing Arp3 Thr293 with Gln to mimic Met oxidation promotes network disassembly. Thus, MICAL2 regulates a subset of Arp2/3 complexes to control branched actin network disassembly.

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