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At least 577 records · Page 32Linked to original sources
PubMed1994

Comparative in vitro phagocytosis and F-actin polymerization of bovine neonatal neutrophils.

Analysis of neonatal neutrophil (PMN) functions should help to reveal factors which could contribute to the impaired host defense system of neonates. We analysed functional parameters of PMN from newborn calves (N-PMN) and adult bovines (A-PMN): cellular volume and F-actin content upon stimulation with complement factors, by cytofluorometry and phagocytosis of E. coli 78:80B with a colorimetric assay. Polymerization of F-actin was rapid in both N- and A-PMN, but reached higher levels in N-PMN. N-PMN are significantly smaller than A-PMN throughout the whole activation time. Percentage of phagocytosing PMN, the rate of phagocytosis, and the rate of killing are similar between A- and N-PMN after opsonization of bacteria with adult serum (AS). Opsonization with newborn serum (NS) reduced all three examined parameters: in A-PMN more (P < 0.001) than in N-PMN (P < 0.05). However, when phagocytosis was compared following age-matched opsonization (N-PMN and NS; A-PMN and AS), N-PMN phagocytosed less (P < 0.001) bacteria per PMN than A-PMN. Additionally, steroidal (dexamethasone) and non-steroidal (phenylbutazone) anti-inflammatory drugs inhibited phagocytosis by N-PMN less than by A-PMN. Higher relative F-actin content of N-PMN can be correlated with the documented functional hyperactivity of bovine N-PMN. However, the exaggerated impairment of phagocytosis in calves observed after age-matched opsonization of bacteria could potentially indicate a specific host defence defect.

Actins↗
PubMed1995

Diminished actin polymerization of neonatal neutrophils determined by a microvolume whole blood method.

To investigate the actin response to chemotactic stimulation of neonatal neutrophils, we developed a new procedure to measure neutrophil F-actin content directly in microvolume whole blood (100 microliters sample each test). Using this procedure, we compared neutrophil actin response to N-formyl-methyonyl-leucyl-phenylalanine (FMLP) between groups of normal neonate and adult volunteers. Relative F-actin content (FMLP/control ratio) of neonatal neutrophils is significantly lower than those of adult volunteers' neutrophils both at 30 sec (1.94 +/- 0.34 vs. 2.25 +/- 0.31; n = 16, p < 0.05, t test) and 60 sec (2.23 +/- 0.19 vs. 2.40 +/- 0.27; n = 16, p < 0.05, t test) after FMLP stimulation. These results provide new evidence of impairment of neutrophil actin response in the neonate, which may partially explain the observed chemotactic defect in neonatal neutrophils.

Actins↗
PubMed1987

Entry of Shigella flexneri into HeLa cells: evidence for directed phagocytosis involving actin polymerization and myosin accumulation.

The enteroinvasive bacterium Shigella flexneri expresses a plasmid-mediated capacity to penetrate into nonphagocytic cells. By using 7-nitrobenz-2-oxa-1,3-diazole-phallacidin (NBD-phallacidin), a fluorescent dye which specifically stains microfilaments, we observed condensations of filamentous actin underneath the plasma membrane of HeLa cells which interacted with the invasive isolate M90T. As demonstrated by indirect immunofluorescence with the antimyosin monoclonal antibody CC-212, myosin accumulated at the same sites. The entry process could be synchronized by using strain SC301, a pIL22 transformant of M90T. pIL22, a recombinant plasmid encoding the Escherichia coli afimbrial adhesin AFA I, rendered shigellae highly adherent to HeLa cells. Using such a system, we demonstrated that the occurrence of bacterial penetration and the appearance of structures brightly stained by NBD-phallacidin were simultaneous events. Such microfilamentous structures resulted from de novo polymerization of the monomeric actin pool in a DNase I inhibition assay, as shown by measurement of the monomeric versus total actin content of infected HeLa cells. These data provide direct evidence that the penetration of S. flexneri into HeLa cells occurs through a mechanism similar to phagocytosis by professional phagocytes.

Actin Cytoskeleton↗
PubMed2000

Profilin enhances Cdc42-induced nucleation of actin polymerization.

We find that profilin contributes in several ways to Cdc42-induced nucleation of actin filaments in high speed supernatant of lysed neutrophils. Depletion of profilin inhibited Cdc42-induced nucleation; re-addition of profilin restored much of the activity. Mutant profilins with a decreased affinity for either actin or poly-l-proline were less effective at restoring activity. Whereas Cdc42 must activate Wiskott-Aldrich Syndrome protein (WASP) to stimulate nucleation by the Arp2/3 complex, VCA (verpolin homology, cofilin, and acidic domain contained in the COOH-terminal fragment of N-WASP) constitutively activates the Arp2/3 complex. Nucleation by VCA was not inhibited by profilin depletion. With purified N-WASP and Arp2/3 complex, Cdc42-induced nucleation did not require profilin but was enhanced by profilin, wild-type profilin being more effective than mutant profilin with reduced affinity for poly-l-proline. Nucleation by the Arp2/3 complex is a function of the free G-actin concentration. Thus, when profilin addition decreased the free G-actin concentration, it inhibited Cdc42- and VCA-induced nucleation. However, when profilin was added with G-actin in a ratio that maintained the initial free G-actin concentration, it increased the rate of both Cdc42- and VCA-induced nucleation. This enhancement, also seen with purified proteins, was greatest when the free G-actin concentration was low. These data suggest that under conditions present in intact cells, profilin enhances nucleation by activated Arp2/3 complex.

Actins↗
PubMed2003

A balance of capping protein and profilin functions is required to regulate actin polymerization in Drosophila bristle.

Profilin is a well-characterized protein known to be important for regulating actin filament assembly. Relatively few studies have addressed how profilin interacts with other actin-binding proteins in vivo to regulate assembly of complex actin structures. To investigate the function of profilin in the context of a differentiating cell, we have studied an instructive genetic interaction between mutations in profilin (chickadee) and capping protein (cpb). Capping protein is the principal protein in cells that caps actin filament barbed ends. When its function is reduced in the Drosophila bristle, F-actin levels increase and the actin cytoskeleton becomes disorganized, causing abnormal bristle morphology. chickadee mutations suppress the abnormal bristle phenotype and associated abnormalities of the actin cytoskeleton seen in cpb mutants. Furthermore, overexpression of profilin in the bristle mimics many features of the cpb loss-of-function phenotype. The interaction between cpb and chickadee suggests that profilin promotes actin assembly in the bristle and that a balance between capping protein and profilin activities is important for the proper regulation of F-actin levels. Furthermore, this balance of activities affects the association of actin structures with the membrane, suggesting a link between actin filament dynamics and localization of actin structures within the cell.

Actin Depolymerizing Factors↗
PubMed1985

Structure-activity relationship of thirty-nine cytochalasans observed in the effects on cellular structures and cellular events and on actin polymerization in vitro.

The effects of twenty-three natural cytochalasans and sixteen synthetic derivatives on actin-distribution and alteration in morphology of C3H-2K cells, inhibition of lymphocyte capping, and inhibition of actin filament elongation were compared. The effects on cellular level and the in vitro effects showed positive correlation and the structure-activity relationship observed is discussed.

Actins↗
PubMed1992

Cytochalasins induce actin polymerization in human leukocytes.

We studied the effect of cytochalasins (B, D, and E) on the F-actin content in human neutrophils and lymphocytes using NBD-phallacidin labeling followed by flow cytometry. All three cytochalasins induced a concentration- and time-dependent increase in the F-actin content in both cell types. The order of potency was cytochalasin D greater than E greater than B. The increase in F-actin content was accompanied by a decrease in the G-actin content as measured by DNase I inhibition assay. These observations suggest that in intact cells cytochalasins may function differently compared to purified and semipurified systems, and their effects may be modified through other actin-binding or sequestering proteins. 2-deoxyglucose (20 mM) caused a decrease in the basal F-actin content and significantly reduced the change induced by the cytochalasins. These results suggest that the state of actin in intact cells is regulated by cytosolic ATP levels, primarily by the integrity of the glycolytic pathway. Based on these observations, we conclude that the mechanism of action of cytochalasins in intact cells is more complex than current models suggest.

Actin Cytoskeleton↗
PubMed2005

The NF2 tumor suppressor Merlin and the ERM proteins interact with N-WASP and regulate its actin polymerization function.

The function of the NF2 tumor suppressor merlin has remained elusive despite increasing evidence for its role in actin cytoskeleton reorganization. The closely related ERM proteins (ezrin, radixin, and moesin) act as linkers between the cell membrane and cytoskeleton, and have also been implicated as active actin reorganizers. We report here that merlin and the ERMs can interact with and regulate N-WASP, a critical regulator of actin dynamics. Merlin and moesin were found to inhibit N-WASP-mediated actin assembly in vitro, a function that appears independent of their ability to bind actin. Furthermore, exogenous expression of a constitutively active ERM inhibits N-WASP-dependent Shigella tail formation, suggesting that the ERMs may function as inhibitors of N-WASP function in vivo. This novel function of merlin and the ERMs illustrates a mechanism by which these proteins directly exert their effects on actin reorganization and also provides new insight into N-WASP regulation.

Actins↗
PubMed2002

Cell-cell adhesion and RhoA-mediated actin polymerization are independent phenomena in microtubule disrupted keratinocytes.

E-cadherin-mediated adherens junction formation and maintenance are thought to involve actin filament rearrangements through the action of small GTPases. Recently, we demonstrated that microtubule disruption in normal human epidermal keratinocytes grown in low calcium media conditions induces cell-cell adhesion by redistribution of endogenous E-cadherin, and it promotes stress fiber formation. This actin rearrangement was apparently mediated by RhoA activation. This model system therefore provides a tool with which to dissect relationships between cell-cell adhesion and Rho-mediated stress fiber formation. In this study, we have demonstrated in normal human epidermal keratinocytes that disruption of actin structures including stress fibers does not interfere with E-cadherin redistribution during microtubule-induced cell-cell adhesion. Moreover, this cell-cell adhesion could not be blocked by RhoA inactivation at the level for inhibition of stress fiber formation. Additionally, in the immortalized HaCaT keratinocyte cell line, which does not undergo cell-cell adhesion after microtubule disruption in low calcium conditions, expression of dominant-active RhoA could induce stress fiber formation without inducing adhesion. On the other hand, a variant of the HaCaT cell line, HC-R1, showed microtubule-disruption-induced cell-cell adhesion without stress fiber formation. Together, our results suggest that, in keratinocytes, the process of cell adhesion can occur independently of RhoA-mediated stress fiber formation.

Actins↗