Unpolymerized actin in fibroblasts and brain

Bray, Dennis; Thomas, Clive

doi:10.1016/0022-2836(76)90233-3

Cited by 178 publications

(83 citation statements)

References 18 publications

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“…Such structured nontilamentous actin may affect our understanding of actin assembly in several ways. For example, it is possible that the bead structures may enter the insoluble fraction following cell lysis, thus assays of cellular actin polymerization based on sedimentation may yield an overestimate of filamentous actin (Bray and Thomas, 1976). Since the structured nonfilamentous actin may not inhibit DNase I activities, assays based on DNase I may also be affected (Blikstad et al, 1978).…”

Section: Discussionmentioning

confidence: 99%

“…At least some of these processes, most notably cell locomotion, have been shown to involve the de novo polymerization of actin filaments (Wang, 1985;Theriot and Mitchison, 1991). Since it is known that cultured cells maintain a high concentration of unpolymerized actin (Bray and Thomas, 1976;Blikstad et al, 1978), through association with various monomer sequestering factors (Cooper, 1991;Hartwig and Kwiatkowski, 1991;Safer, 1992;Nachmias, 1993), the polymerization of new filaments presumably requires the release of sequestered actin subunits followed by the assembly of subunits onto nucleation sites or filament ends. Therefore, to understand the mechanism of the regulation of aetin assembly, it is crucial to gain information concerning the distribution of nonfilamentous actin, the regulation of sequestration factors, and the activity of assembly sites.…”

mentioning

confidence: 99%

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Localization and dynamics of nonfilamentous actin in cultured cells [published erratum appears in J Cell Biol 1993 Nov;123(3):following 767]

Cao¹

1993

The Journal of Cell Biology

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Abstract. Although the distribution of filamentous actin is well characterized in many cell types, the distribution of nonfilamentous actin remains poorly understood. To determine the relative distribution of filamentous and nonfilamentous actin in cultured NRK cells, we have used a number of labeling agents that differ with respect to their specificities toward the filamentous or nonfilamentous form, including monoclonal and polyclonal anti-actin antibodies, vitamin D-binding protein (DBP), and fluorescent phalloidin. Numerous punctate structures were identified that bind poorly to phalloidin but stain positively with several anti-actin antibodies. These bead structures also stain with DBP, suggesting that they are enriched in nonfilamentous actin. Similar punctate structures were observed after the microinjection of fluorescently labeled actin into living cells, allowing us to examine their dynamics in living cells. The actin-containing punctate structures were observed predominantly in the region behind lamellipodia, particularly in spreading cells induced by wounding confluent monolayers. Time-lapse recording of cells injected with fluorescent actin indicated that they form continuously near the leading edge and move centripetally toward the nucleus. Our results suggest that at least part of the unpolymerized actin molecules are localized at discrete sites, possibly as complexes with monomer sequestering proteins. These structures may represent transient storage sites of G-actin within the cell which can be transformed rapidly into actin filaments upon stimulation by specific signals.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Localization and dynamics of nonfilamentous actin in cultured cells [published erratum appears in J Cell Biol 1993 Nov;123(3):following 767]

Cao¹

1993

The Journal of Cell Biology

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“…Purification of ␤-Actin and ARF1-␤-actin was purified from rat brain as described previously (28) and assessed to be Ͼ90% pure by Coomassie staining. Myristoylated recombinant ARF1 was expressed in Escherichia coli and purified (29).…”

Section: Identification Of a 43-kda Protein Using Peptide Mass Fingermentioning

confidence: 99%

Actin Directly Interacts with Phospholipase D, Inhibiting Its Activity

Lee

Park

Kim

et al. 2001

Journal of Biological Chemistry

105

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Mammalian phospholipase D (PLD) plays a key role in several signal transduction pathways and is involved in many diverse functions. To elucidate the complex molecular regulation of PLD, we investigated PLD-binding proteins obtained from rat brain extract. Here we report that a 43-kDa protein in the rat brain, ␤-actin, acts as a major PLD2 direct-binding protein as revealed by peptide mass fingerprinting in combination with matrixassisted laser desorption ionization/time-of-flight mass spectrometry. We also determined that the region between amino acids 613 and 723 of PLD2 is required for the direct binding of ␤-actin, using bacterially expressed glutathione S-transferase fusion proteins of PLD2 fragments. Intriguingly, purified ␤-actin potently inhibited both phosphatidylinositol-4,5-bisphosphateand oleate-dependent PLD2 activities in a concentrationdependent manner (IC 50 ‫؍‬ 5 nM). In a previous paper, we reported that ␣-actinin inhibited PLD2 activity in an interaction-dependent and an ADP-ribosylation factor 1 In vitro binding analyses showed that ␤-actin could displace ␣-actinin binding to PLD2, demonstrating independent interaction between cytoskeletal proteins and PLD2. Furthermore, ARF1 could steer the PLD2 activity in a positive direction regardless of the inhibitory effect of ␤-actin on PLD2. We also observed that ␤-actin regulates PLD1 and PLD2 with similar binding and inhibitory potencies. Immunocytochemical and co-immunoprecipitation studies demonstrated the in vivo interaction between the two PLD isozymes and actin in cells. Taken together, these results suggest that the regulation of PLD by cytoskeletal proteins, ␤-actin and ␣-actinin, and ARF1 may play an important role in cytoskeleton-related PLD functions. Mammalian phospholipase D (PLD)1 hydrolyzes phosphatidylcholine (PC) to generate phosphatidic acid and choline in response to a variety of signals, which can include hormones, neurotransmitters, and growth factors (1). phosphatidic acid itself has been shown to be an intracellular lipid second messenger and to be involved in multiple physiological events such as the promotion of mitogenesis, stimulation of respiratory bursts, secretory processes, actin cytoskeletal reorganization, and the activation of Raf-1 kinase and phosphatidylinositol 4-phosphate (PtdIns4P) 5-kinase isoforms in a large number of cells. These relationships suggest that agonist-induced PLD activation may play roles in multiple signaling events (2-7). The mammalian PLD isozymes identified thus far, PLD1 and PLD2, share a sequence homology of ϳ50%, but they have very different regulatory properties. PLD1 has low basal activity in the presence of phosphatidylinositol-4,5-bisphosphate (PIP 2 ) and can be activated by several cytosolic factors including protein kinase C ␣ and small GTP-binding proteins such as Rho A, Rac-1, ARF1, RalA, and CDC42 (8 -15). PLD2 also depends on PIP 2 but has a higher basal activity than PLD1 (16), and it has been proposed that PLD2 may be closely associated with different cellular inhibitors. Alth...

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“…Synaptosomes contain both actin and myosin (3,4) as well as a Ca2+-stimulated myosin-like ATPase activity, which is associated with synaptic vesicles (3). Actin has been identified in highly purified synaptic vesicle preparations (12) and is also present, mainly as unpolymerized Gactin, within neuronal cytoplasm (13).…”

mentioning

confidence: 99%

Serotonin binds specifically and saturably to an actin-like protein isolated from rat brain synaptosomes.

Small¹,

Wurtman²

1984

Proc. Natl. Acad. Sci. U.S.A.

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A soluble serotonin-binding protein was identified in a high-speed supernatant fraction of an osmotically shocked rat brain synaptosome (P2) preparation. The binding of serotonin was saturable (B. = 6.0 umol per mg of protein) and was specific for serotonin and a few structurally related compounds including dopamine and norepinephrine. Binding of serotonin (1 ,uM) was inhibited "40% by chlorpromazine (10 AuM). The affinity of serotonin for the binding protein was low in the crude extract (Kd = 1.7 X 10-3 M). However, on purification by chromatography on a column of phenothiazine agarose, a higher affinity (Kd = 10-5 M) binding component was also observed. The purified protein was greatly enriched in a polypeptide Of Mr of 43,000 that comigrated on polyacrylamide gel with skeletal muscle actin. Muscle actin also bound serotonin, and the binding to actin was similar to that of the purified protein in both the specificity of the binding and the affinity for serotonin. It is likely that the serotoninbinding protein is identical to cytoplasmic G-actin or an actinlike protein of similar molecular weight.

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Unpolymerized actin in fibroblasts and brain

Cited by 178 publications

References 18 publications

Localization and dynamics of nonfilamentous actin in cultured cells [published erratum appears in J Cell Biol 1993 Nov;123(3):following 767]

Localization and dynamics of nonfilamentous actin in cultured cells [published erratum appears in J Cell Biol 1993 Nov;123(3):following 767]

Actin Directly Interacts with Phospholipase D, Inhibiting Its Activity

Serotonin binds specifically and saturably to an actin-like protein isolated from rat brain synaptosomes.

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