scholarly journals A unified role for membrane-cortex detachment during cell protrusion initiation

2019 ◽  
Author(s):  
Erik S. Welf ◽  
Christopher E. Miles ◽  
Jaewon Huh ◽  
Meghan K. Driscoll ◽  
Tadamoto Isogai ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Force Generation ◽  
Membrane Protrusion ◽  
Cell Morphogenesis ◽  
Quarter Century ◽  
Cell Protrusion ◽  
Optimal Balance ◽  
Membrane Tethering ◽  
Membrane Protrusions ◽  
Intracellular Pressure

AbstractCell morphogenesis employs a diversity of membrane protrusions. They are discriminated by differences in force generation. Actin polymerization is the best studied mechanism of force generation, but growing interest in how variable molecular conditions and microenvironments alter morphogenesis has revealed other mechanisms, including intracellular pressure. Here, we show that local depletion of membrane cortex links is an essential step in the initiation of both pressure-based and actin-based protrusions. This observation challenges the quarter-century old Brownian ratchet model of actin-driven membrane protrusion, which requires an optimal balance of actin filament growth and membrane tethering. An updated model confirms membrane-filament detachment is necessary to activate the ratchet mechanism. These findings unify the regulation of different protrusion types, explaining how cells generate robust yet flexible strategies of morphogenesis.

scholarly journals Optogenetic control of cell morphogenesis on protein micropatterns

2019 ◽  
Author(s):  
Katja Zieske ◽  
R Dyche Mullins
Keyword(s):  
Signaling Pathways ◽  
Adhesion Molecules ◽  
Intracellular Signaling ◽  
Control Cell ◽  
Tissue Formation ◽  
Actin Assembly ◽  
Cell Morphogenesis ◽  
Cell Protrusion ◽  
Cytoskeletal Dynamics ◽  
Membrane Protrusions

AbstractCell morphogenesis is critical for embryonic development, tissue formation, and wound healing. Our ability to manipulate endogenous mechanisms to control cell shape, however, remains limited. Here we combined surface micropatterning of adhesion molecules with optogenetic activation of intracellular signaling pathways to control the nature and morphology of cellular protrusions. We employed geometry-dependent pre-organization of cytoskeletal structures together with acute activation of signaling pathways that control actin assembly to create a tool capable of generating membrane protrusions at defined cellular locations. Further, we find that the size of microfabricated patterns of adhesion molecules influences the molecular mechanism of cell protrusion: larger patterns enable cells to create actin-filled lamellipodia while smaller patterns promote formation of spherical blebs. Optogenetic perturbation of signaling pathways in these cells changes the size of blebs and convert them into lamellipodia. Our results demonstrate how the coordinated manipulation of adhesion geometry and cytoskeletal dynamics can be used to control membrane protrusion and cell morphogenesis.

scholarly journals Proteolysis of Cortactin by Calpain Regulates Membrane Protrusion during Cell Migration

2006 ◽  
Vol 17 (1) ◽  
pp. 239-250 ◽  
Author(s):  
Benjamin J. Perrin ◽  
Kurt J. Amann ◽  
Anna Huttenlocher
Keyword(s):  
Cell Migration ◽  
Actin Polymerization ◽  
Biochemical Properties ◽  
Actin Binding ◽  
Potential Candidate ◽  
Membrane Protrusion ◽  
Cortical Actin ◽  
Membrane Protrusions ◽  
Src Homology ◽  
Calpain 2

Calpain 2 regulates membrane protrusion during cell migration. However, relevant substrates that mediate the effects of calpain on protrusion have not been identified. One potential candidate substrate is the actin binding protein cortactin. Cortactin is a Src substrate that drives actin polymerization by activating the Arp2/3 complex and also stabilizes the cortical actin network. We now provide evidence that proteolysis of cortactin by calpain 2 regulates membrane protrusion dynamics during cell migration. We show that cortactin is a calpain 2 substrate in fibroblasts and that the preferred cleavage site occurs in a region between the actin binding repeats and the α-helical domain. We have generated a mutant cortactin that is resistant to calpain proteolysis but retains other biochemical properties of cortactin. Expression of the calpain-resistant cortactin, but not wild-type cortactin, impairs cell migration and increases transient membrane protrusion, suggesting that calpain proteolysis of cortactin limits membrane protrusions and regulates migration in fibroblasts. Furthermore, the enhanced protrusion observed with the calpain-resistant cortactin requires both the Arp2/3 binding site and the Src homology 3 domain of cortactin. Together, these findings suggest a novel role for calpain-mediated proteolysis of cortactin in regulating membrane protrusion dynamics during cell migration.

scholarly journals Membrane proximal F-actin restricts local membrane protrusions and directs cell migration

2019 ◽  
Author(s):  
Anjali Bisaria ◽  
Arnold Hayer ◽  
Damien Garbett ◽  
Daniel Cohen ◽  
Tobias Meyer
Keyword(s):  
Plasma Membrane ◽  
Cell Migration ◽  
Actin Polymerization ◽  
Cell Polarization ◽  
Structural Parameter ◽  
Low Density ◽  
Membrane Protrusion ◽  
Fluorescent Reporter ◽  
Membrane Protrusions ◽  
Actin Concentration

AbstractA major component of cell migration is F-actin polymerization driven membrane protrusion in the front. However, F-actin proximal to the plasma membrane also has a scaffolding role to support and attach the membrane. Here we developed a fluorescent reporter to monitor changes in the density of membrane proximal F-actin during membrane protrusion and cell migration. Strikingly, unlike total F-actin concentration, which is high in the front of migrating cells, the density of membrane proximal F-actin is low in the front and high in the back. Furthermore, local membrane protrusions only form following local decreases in membrane proximal F-actin density. Our study suggests that low density of membrane proximal F-actin is a fundamental structural parameter that locally directs membrane protrusions and globally stabilizes cell polarization during cell migration.One Sentence SummaryMembrane protrusion and cell migration are directed by local decreases in the density of membrane proximal F-actin

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 Identification of a 521-Kilodalton Protein (Gli521) Involved in Force Generation or Force Transmission for Mycoplasma mobile Gliding

2005 ◽  
Vol 187 (10) ◽  
pp. 3502-3510 ◽  
Author(s):  
Shintaro Seto ◽  
Atsuko Uenoyama ◽  
Makoto Miyata
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Conformational Changes ◽  
Peptide Bond ◽  
Gliding Motility ◽  
Force Generation ◽  
Mycoplasma Species ◽  
Membrane Protrusion ◽  
Force Transmission ◽  
Inhibitory Effects

ABSTRACT Several mycoplasma species are known to glide on solid surfaces such as glass in the direction of the membrane protrusion, but the mechanism underlying this movement is unknown. To identify a novel protein involved in gliding, we raised monoclonal antibodies against a detergent-insoluble protein fraction of Mycoplasma mobile, the fastest glider, and screened the antibodies for inhibitory effects on gliding. Five monoclonal antibodies stopped the movement of gliding mycoplasmas, keeping them on the glass surface, and all of them recognized a large protein in immunoblotting. This protein, named Gli521, is composed of 4,738 amino acids, has a predicted molecular mass of 520,559 Da, and is coded downstream of a gene for another gliding protein, Gli349, which is known to be responsible for glass binding during gliding. Edman degradation analysis indicated that the N-terminal region is processed at the peptide bond between the amino acid residues at positions 43 and 44. Analysis of gliding mutants isolated previously revealed that the Gli521 protein is missing in a nonbinding mutant, m9, where the gli521 gene is truncated by a nonsense mutation at the codon for the amino acid at position 1170. Immunofluorescence and immunoelectron microscopy indicated that Gli521 localizes all around the base of the membrane protrusion, at the “neck,” as previously observed for Gli349. Analysis of the inhibitory effects of the anti-Gli521 antibody on gliding motility revealed that this protein is responsible for force generation or force transmission, a role distinct from that of Gli349, and also suggested conformational changes of Gli349 and Gli521 during gliding.

scholarly journals Kindlin-2 recruits paxillin and Arp2/3 to promote membrane protrusions during initial cell spreading

2017 ◽  
Vol 216 (11) ◽  
pp. 3785-3798 ◽  
Author(s):  
Ralph T. Böttcher ◽  
Maik Veelders ◽  
Pascaline Rombaut ◽  
Jan Faix ◽  
Marina Theodosiou ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Adhesion ◽  
Adaptor Protein ◽  
Cell Spreading ◽  
Dual Role ◽  
Membrane Protrusion ◽  
Pleckstrin Homology ◽  
Initial Cell ◽  
Membrane Protrusions ◽  
Lamellipodia Formation

Cell spreading requires the coupling of actin-driven membrane protrusion and integrin-mediated adhesion to the extracellular matrix. The integrin-activating adaptor protein kindlin-2 plays a central role for cell adhesion and membrane protrusion by directly binding and recruiting paxillin to nascent adhesions. Here, we report that kindlin-2 has a dual role during initial cell spreading: it binds paxillin via the pleckstrin homology and F0 domains to activate Rac1, and it directly associates with the Arp2/3 complex to induce Rac1-mediated membrane protrusions. Consistently, abrogation of kindlin-2 binding to Arp2/3 impairs lamellipodia formation and cell spreading. Our findings identify kindlin-2 as a key protein that couples cell adhesion by activating integrins and the induction of membrane protrusions by activating Rac1 and supplying Rac1 with the Arp2/3 complex.

scholarly journals Dynamic microtubules regulate cellular contractility during T-cell activation

2017 ◽  
Vol 114 (21) ◽  
pp. E4175-E4183 ◽  
Author(s):  
King Lam Hui ◽  
Arpita Upadhyaya
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Actin Polymerization ◽  
Cell Activation ◽  
Adaptive Immune Response ◽  
Cell Receptor ◽  
Force Generation ◽  
Actin Dynamics ◽  
Bipolar Filament

T-cell receptor (TCR) triggering and subsequent T-cell activation are essential for the adaptive immune response. Recently, multiple lines of evidence have shown that force transduction across the TCR complex is involved during TCR triggering, and that the T cell might use its force-generation machinery to probe the mechanical properties of the opposing antigen-presenting cell, giving rise to different signaling and physiological responses. Mechanistically, actin polymerization and turnover have been shown to be essential for force generation by T cells, but how these actin dynamics are regulated spatiotemporally remains poorly understood. Here, we report that traction forces generated by T cells are regulated by dynamic microtubules (MTs) at the interface. These MTs suppress Rho activation, nonmuscle myosin II bipolar filament assembly, and actin retrograde flow at the T-cell–substrate interface. Our results suggest a novel role of the MT cytoskeleton in regulating force generation during T-cell activation.

scholarly journals The small GTPase Cdc42 promotes membrane protrusion during polar body emission via ARP2-nucleated actin polymerization

2011 ◽  
Vol 17 (5) ◽  
pp. 305-316 ◽  
Author(s):  
J. Leblanc ◽  
X. Zhang ◽  
D. McKee ◽  
Z.- B. Wang ◽  
R. Li ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Polar Body ◽  
Small Gtpase ◽  
Membrane Protrusion

scholarly journals ADP ribosylation factor 6 is activated and controls membrane delivery during phagocytosis in macrophages

2003 ◽  
Vol 161 (6) ◽  
pp. 1143-1150 ◽  
Author(s):  
Florence Niedergang ◽  
Emma Colucci-Guyon ◽  
Thierry Dubois ◽  
Graça Raposo ◽  
Philippe Chavrier
Keyword(s):  
Actin Polymerization ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Adp Ribosylation ◽  
Transient Activation ◽  
Endosomal Recycling ◽  
Membrane Protrusions ◽  
Specific Receptors ◽  
Intracellular Vesicles ◽  
Adp Ribosylation Factor

Engulfment of particles by phagocytes is induced by their interaction with specific receptors on the cell surface, which leads to actin polymerization and the extension of membrane protrusions to form a closed phagosome. Membrane delivery from internal pools is considered to play an important role in pseudopod extension during phagocytosis. Here, we report that endogenous ADP ribosylation factor 6 (ARF6), a small GTP-binding protein, undergoes a sharp and transient activation in macrophages when phagocytosis was initiated via receptors for the Fc portion of immunoglobulins (FcRs). A dominant-negative mutant of ARF6 (T27N mutation) dramatically affected FcR-mediated phagocytosis. Expression of ARF6-T27N lead to a reduction in the focal delivery of vesicle-associated membrane protein 3+ endosomal recycling membranes at phagocytosis sites, whereas actin polymerization was unimpaired. This resulted in an early blockade in pseudopod extension and accumulation of intracellular vesicles, as observed by electron microscopy. We conclude that ARF6 is a major regulator of membrane recycling during phagocytosis.

Thromboxane-induced actin polymerization in hypoxic pulmonary artery is independent of Rho

2012 ◽  
Vol 302 (1) ◽  
pp. L13-L26 ◽  
Author(s):  
Jena Fediuk ◽  
Alexey Gutsol ◽  
Nora Nolette ◽  
Shyamala Dakshinamurti
Keyword(s):  
Rho Gtpases ◽  
Laser Scanning ◽  
Actin Polymerization ◽  
Isometric Force ◽  
Renal Arteries ◽  
Force Generation ◽  
Lim Kinase ◽  
Thromboxane Receptor ◽  
Laser Scanning Cytometry ◽  
And Control

Actin polymerization (APM), regulated by Rho GTPases, promotes myocyte force generation. Hypoxia is known to impede postnatal disassembly of the actin cytoskeleton in pulmonary arterial (PA) myocytes. We compared basal and agonist-induced APM in myocytes from PA and descending aorta (Ao), under hypoxic and normoxic conditions. We also examined effects of thromboxane challenge on force generation and cytoskeletal assembly in resistance PA and renal arteries from neonatal swine with persistent pulmonary hypertension (PPHN) induced by 72-h normobaric hypoxia, compared with age-matched controls. Synthetic and contractile phenotype myocytes from neonatal porcine PA or Ao were grown in hypoxia (10% O2) or normoxia (21% O2) for 7 days, then challenged with 10−6 M thromboxane mimetic U46619. F/G actin ratio was quantified by laser-scanning cytometry and by cytoskeletal fractionation. Thromboxane receptor (TP) G protein coupling was measured by immunoprecipitation and probing for Gαq, G12, or G13, RhoA activation by Rhotekin-RBD affinity precipitation, and LIM kinase (LIMK) and cofilin phosphorylation by Western blot. Isometric force to serial concentrations of U46619 was measured in muscular pulmonary and renal arteries from PPHN and control swine; APM was quantified in fixed contracted vessels. Contractile PA myocytes exhibit marked Rho-dependent APM in hypoxia, with increased active RhoA and LIMK phosphorylation. Their additional APM response to U46619 challenge is independent of RhoA, reflecting decreased TP association with G12/13 in favor of Gαq. In contrast, hypoxic contractile Ao myocytes polymerize actin modestly and depolymerize to U46619. Both basal APM and the APM response to U46619 are increased in PPHN PA. APM corresponds with increased force generation to U46619 challenge in PPHN PA but not renal arteries.

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