scholarly journals Mechanisms responsible for F-actin stabilization after lysis of polymorphonuclear leukocytes.

1992 ◽  
Vol 116 (5) ◽  
pp. 1123-1134 ◽  
Author(s):  
M L Cano ◽  
L Cassimeris ◽  
M Fechheimer ◽  
S H Zigmond
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Actin Polymerization ◽  
Cell Movement ◽  
Actin Binding ◽  
Cross Linking ◽  
Macromolecular Complex ◽  
Actin Depolymerization ◽  
Gravitational Forces ◽  
End Capping

While actin polymerization and depolymerization are both essential for cell movement, few studies have focused on actin depolymerization. In vivo, depolymerization can occur exceedingly rapidly and in a spatially defined manner: the F-actin in the lamellipodia depolymerizes in 30 s after chemoattractant removal (Cassimeris, L., H. McNeill, and S. H. Zigmond. 1990. J. Cell Biol. 110:1067-1075). To begin to understand the regulation of F-actin depolymerization, we have examined F-actin depolymerization in lysates of polymorphonuclear leukocytes (PMNs). Surprisingly, much of the cell F-actin, measured with a TRITC-phalloidin-binding assay, was stable after lysis in a physiological salt buffer (0.15 M KCl): approximately 50% of the F-actin did not depolymerize even after 18 h. This stable F-actin included lamellar F-actin which could still be visualized one hour after lysis by staining with TRITC-phalloidin and by EM. We investigated the basis for this stability. In lysates with cell concentrations greater than 10(7) cells/ml, sufficient globular actin (G-actin) was present to result in a net increase in F-actin. However, the F-actin stability was not solely because of the presence of free G-actin since addition of DNase I to the lysate did not increase the F-actin loss. Nor did it appear to be because of barbed end capping factors since cell lysates provided sites for barbed end polymerization of exogenous added actin. The stable F-actin existed in a macromolecular complex that pelleted at low gravitational forces. Increasing the salt concentration of the lysis buffer decreased the amount of F-actin that pelleted at low gravitational forces and increased the amount of F-actin that depolymerized. Various actin-binding and cross-linking proteins such as tropomyosin, alpha-actinin, and actin-binding protein pelleted with the stable F-actin. In addition, we found that alpha-actinin, a filament cross-linking protein, inhibited the rate of pyrenyl F-actin depolymerization. These results suggested that actin cross-linking proteins may contribute to the stability of cellular actin after lysis. The activity of crosslinkers may be regulated in vivo to allow rapid turnover of lamellipodia F-actin.

scholarly journals Microtubule Actin Cross-Linking Factor (Macf)

1999 ◽  
Vol 147 (6) ◽  
pp. 1275-1286 ◽  
Author(s):  
Conrad L. Leung ◽  
Dongming Sun ◽  
Min Zheng ◽  
David R. Knowles ◽  
Ronald K.H. Liem
Keyword(s):  
Calcium Binding ◽  
Coiled Coil ◽  
Specific Protein ◽  
Binding Domain ◽  
Full Length ◽  
Actin Binding ◽  
Cross Linking ◽  
Actin Binding Domain

We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends–PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH2 terminus. However, unlike dystonin, mACF7 does not contain a coiled–coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest–specific protein, Gas2. In this paper, we demonstrate that the NH2-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

scholarly journals Thymosin beta 4 sequesters the majority of G-actin in resting human polymorphonuclear leukocytes.

1992 ◽  
Vol 119 (5) ◽  
pp. 1261-1270 ◽  
Author(s):  
L Cassimeris ◽  
D Safer ◽  
V T Nachmias ◽  
S H Zigmond
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Actin Polymerization ◽  
Large Fraction ◽  
Reverse Phase Hplc ◽  
Thymosin Beta 4 ◽  
Rapid Changes ◽  
Human Pmns ◽  
Human Polymorphonuclear Leukocytes ◽  
Kinetics Of

Thymosin beta 4 (T beta 4), a 5-kD peptide which binds G-actin and inhibits its polymerization (Safer, D., M. Elzinga, and V. T. Nachmias. 1991. J. Biol. Chem. 266:4029-4032), appears to be the major G-actin sequestering protein in human PMNs. In support of a previous study by Hannappel, E., and M. Van Kampen (1987. J. Chromatography. 397:279-285), we find that T beta 4 is an abundant peptide in these cells. By reverse phase HPLC of perchloric acid supernatants, human PMNs contain approximately 169 fg/cell +/- 90 fg/cell (SD), corresponding to a cytoplasmic concentration of approximately 149 +/- 80.5 microM. On non-denaturing polyacrylamide gels, a large fraction of G-actin in supernatants prepared from resting PMNs has a mobility similar to the G-actin/T beta 4 complex. Chemoattractant stimulation of PMNs results in a decrease in this G-actin/T beta 4 complex. To determine whether chemoattractant induced actin polymerization results from an inactivation of T beta 4, the G-actin sequestering activity of supernatants prepared from resting and chemoattractant stimulated cells was measured by comparing the rates of pyrenyl-actin polymerization from filament pointed ends. Pyrenyl actin polymerization was inhibited to a greater extent in supernatants from stimulated cells and these results are qualitatively consistent with T beta 4 being released as G-actin polymerizes, with no chemoattractant-induced change in its affinity for G-actin. The kinetics of bovine spleen T beta 4 binding to muscle pyrenyl G-actin are sufficiently rapid to accommodate the rapid changes in actin polymerization and depolymerization observed in vivo in response to chemoattractant addition and removal.

scholarly journals Overexpression of cofilin stimulates bundling of actin filaments, membrane ruffling, and cell movement in Dictyostelium.

1996 ◽  
Vol 132 (3) ◽  
pp. 335-344 ◽  
Author(s):  
H Aizawa ◽  
K Sutoh ◽  
I Yahara
Keyword(s):  
Actin Filaments ◽  
Cell Movement ◽  
Structure And Function ◽  
Cross Linking ◽  
Low Molecular Weight ◽  
Filamentous Actin ◽  
Membrane Ruffling ◽  
And Function

Cofilin is a low molecular weight actin-modulating protein whose structure and function are conserved among eucaryotes. Cofilin exhibits in vitro both a monomeric actin-sequestering activity and a filamentous actin-severing activity. To investigate in vivo functions of cofilin, cofilin was overexpressed in Dictyostelium discoideum cells. An increase in the content of D. discoideum cofilin (d-cofilin) by sevenfold induced a co-overproduction of actin by threefold. In cells over-expressing d-cofilin, the amount of filamentous actin but not that of monomeric actin was increased. Overexpressed d-cofilin co-sedimented with actin filaments, suggesting that the sequestering activity of d-cofilin is weak in vivo. The overexpression of d-cofilin increased actin bundles just beneath ruffling membranes where d-cofilin was co-localized. The overexpression of d-cofilin also stimulated cell movement as well as membrane ruffling. We have demonstrated in vitro that d-cofilin transformed latticework of actin filaments cross-linked by alpha-actinin into bundles probably by severing the filaments. D. discoideum cofilin may sever actin filaments in vivo and induce bundling of the filaments in the presence of cross-linking proteins so as to generate contractile systems involved in membrane ruffling and cell movement.

scholarly journals EGF-induced PIP2 hydrolysis releases and activates cofilin locally in carcinoma cells

2007 ◽  
Vol 179 (6) ◽  
pp. 1247-1259 ◽  
Author(s):  
Jacco van Rheenen ◽  
Xiaoyan Song ◽  
Wies van Roosmalen ◽  
Michael Cammer ◽  
Xiaoming Chen ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Carcinoma Cells ◽  
Actin Binding ◽  
Temporal Regulation ◽  
Vitro Binding ◽  
Epidermal Growth ◽  
Membrane Bound ◽  
Regulation Of Actin Cytoskeleton

Lamellipodial protrusion and directional migration of carcinoma cells towards chemoattractants, such as epidermal growth factor (EGF), depend upon the spatial and temporal regulation of actin cytoskeleton by actin-binding proteins (ABPs). It is generally hypothesized that the activity of many ABPs are temporally and spatially regulated by PIP2; however, this is mainly based on in vitro–binding and structural studies, and generally in vivo evidence is lacking. Here, we provide the first in vivo data that directly visualize the spatial and temporal regulation of cofilin by PIP2 in living cells. We show that EGF induces a rapid loss of PIP2 through PLC activity, resulting in a release and activation of a membrane-bound pool of cofilin. Upon release, we find that cofilin binds to and severs F-actin, which is coincident with actin polymerization and lamellipod formation. Moreover, our data provide evidence for how PLC is involved in the formation of protrusions in breast carcinoma cells during chemotaxis and metastasis towards EGF.

scholarly journals Mechanisms for actin reorganization in chemotactic factor-activated polymorphonuclear leukocytes

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2750-2757
Author(s):  
RG Watts ◽  
TH Howard
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Actin Polymerization ◽  
Insoluble Fraction ◽  
Chemotactic Factor ◽  
Actin Dynamics ◽  
Cross Linking ◽  
Actin Reorganization ◽  
Cytoskeletal Structure ◽  
Cross Links ◽  
Human Pmns

Cytoskeletal structure in polymorphonuclear leukocytes (PMNs) is thought to reflect a simple equilibrium between two actin pools (globular [G]- and filamentous [F] actin). Recent description of two distinct F-actin pools in PMNs (Triton-insoluble [stable] and Triton- soluble [labile] F-actin pools) (Watts and Howard, Cell Motil Cytoskeleton, 21:25, 1992) suggest a tripartite equilibrium between these F-actin pools and G-actin and multiple possible mechanisms for polymerization. To study the contribution of each actin pool to actin dynamics in PMNs, changes in actin content of the Triton-soluble and - insoluble F-actin pools and G-actin in chemotactic factor (CTF)- activated PMNs were measured by NBDphallacidin binding and by gel scans of Triton-lysed PMNs. From 0 to 30 seconds after CTF activation, PMNs rapidly increase total (Triton-soluble + Triton-insoluble) F-actin content (maximum = 1.7- +/- 0.10-fold basal at 30 seconds). Concurrent measures of the actin content of individual actin pools (Triton-soluble and -insoluble F-actin and G-actin) show that at all times (0 to 30 seconds) only the Triton-insoluble F-actin pool grows (maximum = 2.81- +/- 0.73-fold basal at 30 seconds), whereas both the Triton-soluble and G-actin pools simultaneously decrease (50% decrease at 30 seconds). Concurrent growth of one F-actin pool (Triton-insoluble) and loss of another F-actin pool (Triton-soluble) emphasize the functional uniqueness of the F-actin pools and can occur only if the Triton- soluble F-actin anneals or cross-links filament-to-filament with the Triton-insoluble fraction or if the Triton-insoluble F-actin pool first depolymerizes to monomer, which is then added to the Triton-insoluble pool. Because from 0 to 30 seconds after FMLP activation G-actin never increases, but, like the Triton-soluble F-actin progressively decreases, the results suggest that F-actin growth results from simultaneous new filament growth by monomer addition to the Triton- insoluble F-actin and cytoskeletal remodelling by Triton-soluble F- actin annealing or cross-linking to Triton-insoluble F-actin. These findings offer important new insights into the mechanism(s) of actin polymerization in CTF-activated human PMNs.

scholarly journals Interactions of tensin with actin and identification of its three distinct actin-binding domains.

1994 ◽  
Vol 125 (5) ◽  
pp. 1067-1075 ◽  
Author(s):  
S H Lo ◽  
P A Janmey ◽  
J H Hartwig ◽  
L B Chen
Keyword(s):  
Amino Acids ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Sh2 Domain ◽  
Actin Binding ◽  
Molar Ratio ◽  
Actin Assembly ◽  
Binding Domains ◽  
Focal Contacts ◽  
End Capping

Tensin, a 200-kD phosphoprotein of focal contacts, contains sequence homologies to Src (SH2 domain), and several actin-binding proteins. These features suggest that tensin may link the cell membrane to the cytoskeleton and respond directly to tyrosine kinase signalling pathways. Here we identify three distinct actin-binding domains within tensin. Recombinant tensin purified after overexpression by a baculovirus system binds to actin filaments with Kd = 0.1 microM, cross-links actin filaments at a molar ratio of 1:10 (tensin/actin), and retards actin assembly by barbed end capping with Kd = 20 nM. Tensin fragments were constructed and expressed as fusion proteins to map domains having these activities. Three regions from tensin interact with actin: two regions composed of amino acids 1 to 263 and 263 to 463, cosediment with F-actin but do not alter the kinetics of actin assembly; a region composed of amino acids 888-989, with sequence homology to insertin, retards actin polymerization. A claw-shaped tensin dimer would have six potential actin-binding sites and could embrace the ends of two actin filaments at focal contacts.

scholarly journals Distinct phospho-forms of cortactin differentially regulate actin polymerization and focal adhesions

2008 ◽  
Vol 295 (5) ◽  
pp. C1113-C1122 ◽  
Author(s):  
Anne E. Kruchten ◽  
Eugene W. Krueger ◽  
Yu Wang ◽  
Mark A. McNiven
Keyword(s):  
Cell Migration ◽  
Focal Adhesion ◽  
Actin Polymerization ◽  
Control Cell ◽  
Focal Adhesions ◽  
Serine Phosphorylation ◽  
Actin Binding ◽  
Actin Assembly

Cortactin is an actin-binding protein that is overexpressed in many cancers and is a substrate for both tyrosine and serine/threonine kinases. Tyrosine phosphorylation of cortactin has been observed to increase cell motility and invasion in vivo, although it has been reported to have both positive and negative effects on actin polymerization in vitro. In contrast, serine phosphorylation of cortactin has been shown to stimulate actin assembly in vitro. Currently, the effects of cortactin serine phosphorylation on cell migration are unclear, and furthermore, how the distinct phospho-forms of cortactin may differentially contribute to cell migration has not been directly compared. Therefore, we tested the effects of different tyrosine and serine phospho-mutants of cortactin on lamellipodial protrusion, actin assembly within cells, and focal adhesion dynamics. Interestingly, while expression of either tyrosine or serine phospho-mimetic cortactin mutants resulted in increased lamellipodial protrusion and cell migration, these effects appeared to be via distinct processes. Cortactin mutants mimicking serine phosphorylation appeared to predominantly affect actin polymerization, whereas mutation of cortactin tyrosine residues resulted in alterations in focal adhesion turnover. Thus these findings provide novel insights into how distinct phospho-forms of cortactin may differentially contribute to actin and focal adhesion dynamics to control cell migration.

scholarly journals Vascular NRP2 triggers PNET angiogenesis by activating the SSH1-cofilin axis

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Xi Luo ◽  
Jiang-yi He ◽  
Jie Xu ◽  
Shao-yi Hu ◽  
Bang-hui Mo ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Umbilical Vein ◽  
Tube Formation ◽  
Actin Binding ◽  
Important Regulator ◽  
Dataset Analysis ◽  
Patient Prognosis ◽  
Umbilical Vein Endothelial Cells ◽  
The Relationship

Abstract Background Angiogenesis is a critical step in the growth of pancreatic neuroendocrine tumors (PNETs) and may be a selective target for PNET therapy. However, PNETs are robustly resistant to current anti-angiogenic therapies that primarily target the VEGFR pathway. Thus, the mechanism of PNET angiogenesis urgently needs to be clarified. Methods Dataset analysis was used to identify angiogenesis-related genes in PNETs. Immunohistochemistry was performed to determine the relationship among Neuropilin 2 (NRP2), VEGFR2 and CD31. Cell proliferation, wound-healing and tube formation assays were performed to clarify the function of NRP2 in angiogenesis. The mechanism involved in NRP2-induced angiogenesis was detected by constructing plasmids with mutant variants and performing Western blot, and immunofluorescence assays. A mouse model was used to evaluate the effect of the NRP2 antibody in vivo, and clinical data were collected from patient records to verify the association between NRP2 and patient prognosis. Results NRP2, a VEGFR2 co-receptor, was positively correlated with vascularity but not with VEGFR2 in PNET tissues. NRP2 promoted the migration of human umbilical vein endothelial cells (HUVECs) cultured in the presence of conditioned medium PNET cells via a VEGF/VEGFR2-independent pathway. Moreover, NRP2 induced F-actin polymerization by activating the actin-binding protein cofilin. Cofilin phosphatase slingshot-1 (SSH1) was highly expressed in NRP2-activating cofilin, and silencing SSH1 ameliorated NRP2-activated HUVEC migration and F-actin polymerization. Furthermore, blocking NRP2 in vivo suppressed PNET angiogenesis and tumor growth. Finally, elevated NRP2 expression was associated with poor prognosis in PNET patients. Conclusion Vascular NRP2 promotes PNET angiogenesis by activating the SSH1/cofilin/actin axis. Our findings demonstrate that NRP2 is an important regulator of angiogenesis and a potential therapeutic target of anti-angiogenesis therapy for PNET.

scholarly journals Autooxidation as a basis for altered function by polymorphonuclear leukocytes

Blood ◽  
1977 ◽  
Vol 50 (2) ◽  
pp. 327-335 ◽  
Author(s):  
RL Baehner ◽  
LA Boxer ◽  
JM Allen ◽  
J Davis
Keyword(s):  
Vitamin E ◽  
Polymorphonuclear Leukocytes ◽  
Cell Movement ◽  
Membrane Lipid ◽  
Alpha Tocopherol ◽  
Directed Movement ◽  
Aerobic Conditions ◽  
Ingestion Rates ◽  
Human Polymorphonuclear Leukocytes

Abstract To investigate the possibility that human polymorphonuclear leukocytes (PMN) elaborate sufficient amounts of hydrogen peroxide (H2O2) and other radicals of reduced oxygen to be autotoxic and retard directed cell movement and phagocytosis, the rate of ingestion of opsonized lipopolysaccharide-paraffin oil particles and movement through Nuclepore filters were studied. Ingestion rates were increased under anaerobic conditions and in normal aerobic conditions in the presence of extracellular catalase but not superoxide dismutase (SOD) or scavengers of singlet oxygen or hydroxyl radicals. Conversely, ingestion rates were decreased when cells were exposed to H2O2 or a superoxide anion (O2-)-H2O2 generating system of xanthine-xanthine oxidase. Catalase, but not SOD, prevented the effect and also enhanced the directed movement of PMN in normal aerobic conditions. PMN from volunteers administered 1600 U/day of the membrane lipid antioxidant alpha-tocopherol were hyperphagocytic but killed Staphylococcus aureus 502A less effectively than controls, suggesting that less H2O2 was available to damage PMN or kill bacteria. H2O2-dependent stimulation of the hexose monophosphate shunt, H2O2 release from phaogytizing PMN, and fluoresceinated concanavalin A cap formation promoted by H2O2 damage to microtubules were all diminished, but the release of O2- from phagocytizing PMN was not diminished in the vitamin E group. These results support the hypothesis that directed movement and phagocytosis by PMN are attenuated by autooxidative damage to the cell membrane by endogenously derived H2O2 and that the administration in vivo of vitamin E may prevent this damage by scavenging H2O2.

scholarly journals Vascular NRP2 triggers PNET angiogenesis by activating the SSH1-cofilin axis

2020 ◽  
Author(s):  
xi luo ◽  
Jiang-yi He ◽  
Jie Xu ◽  
Shao-yi Hu ◽  
Bang-hui Mo ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Umbilical Vein ◽  
Tube Formation ◽  
Actin Binding ◽  
Potential Therapeutic Target ◽  
Important Regulator ◽  
Dataset Analysis ◽  
Umbilical Vein Endothelial Cells ◽  
The Relationship

Abstract Background: Angiogenesis is a critical step in the growth of pancreatic neuroendocrine tumors (PNETs) and may be a selective target for PNET therapy. However, PNETs are robustly resistant to current anti-angiogenic therapies that primarily target the VEGFR pathway. Thus, the mechanism of PNET angiogenesis urgently needs to be clarified.Methods: Dataset analysis was used to identify angiogenesis-related genes in PNETs. Immunohistochemistry was performed to determine the relationship among Neuropilin 2 (NRP2), VEGFR2 and CD31. Cell proliferation, wound-healing and tube formation assays were performed to clarify the function of NRP2 in angiogenesis. The mechanism involved in NRP2-induced angiogenesis was detected by constructing plasmids with mutant variants and performing Western blot, and immunofluorescence assays. A mouse model was used to evaluate the effect of the NRP2 antibody in vivo, and clinical data were collected from patient records to verify the association between NRP2 and patient prognosis.Results: NRP2, a VEGFR2 co-receptor, was positively correlated with vascularity but not with VEGFR2 in PNET tissues. NRP2 promoted the migration of human umbilical vein endothelial cells (HUVECs) cultured in the presence of conditioned medium PNET cells via a VEGF/VEGFR2-independent pathway. Moreover, NRP2 induced F-actin polymerization by activating the actin-binding protein cofilin. Cofilin phosphatase slingshot-1 (SSH1) was highly expressed in NRP2-activating cofilin, and silencing SSH1 ameliorated NRP2-activated HUVEC migration and F-actin polymerization. Furthermore, blocking NRP2 in vivo suppressed PNET angiogenesis and tumor growth. Finally, elevated NRP2 expression was associated with poor prognosis in PNET patients.Conclusion: Vascular NRP2 promotes PNET angiogenesis by activating the SSH1/cofilin/actin axis. Our findings demonstrate that NRP2 is an important regulator of angiogenesis and a potential therapeutic target of anti-angiogenesis therapy for PNET.

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