The Arp2/3 complex regulates many cellular processes by stimulating formation of branched actin filament networks. Because three of its seven subunits exist as two different isoforms, mammals produce a family of Arp2/3 complexes with different properties that may be suited to different physiological contexts. To shed light on how isoform diversification affects Arp2/3 function, we determined a 4.2 Å resolution cryo-EM structure of the most active human Arp2/3 complex containing ARPC1B and ARPC5L, and compared it with the structure of the least active ARPC1A-ARPC5-containing complex. The architecture of each isoformspecified Arp2/3 is the same. Strikingly, however, the N-terminal half of ARPC5L is partially disordered compared to ARPC5, suggesting that this region of ARPC5/ARPC5L is an important determinant of complex activity. Confirming this idea, the nucleation activity of Arp2/3 complexes containing hybrid ARPC5/ARPC5L subunits is higher when the ARPC5L N-terminus is present, thereby explaining activity differences between the different Arp2/3 complexes.

Phosphoinositidase C (PIC) activity is stimulated by a variety of hormones, neurotransmitters and growth factors to generate two second messengers by its action on phosphatidylinositol4,Sbisphosphate. The phospholipid head group, inositol 1,4,5trisphosphate (Ins (1,4,5)P3), when released into the cytosol acts upon specific receptors to release intracellular Ca2+ stores, while the lipid remnant, sn-1 ,Z-diacylglycerol (DAG) remains in the membrane where it is an activator of a number of protein kinase C (PKC) isoforms [I]. It has been proposed, largely on evidence gained from experiments where PKC is acutely activated with phorbol esters, that PKC exerts a negative modulatory influence on PIC, so constituting a short negative feedback loop. These experiments however, do not necessarily indicate the presence of an active feedback loop under normal conditions of agonist stimulation. In this study I have examined the Ins(l,4,5)P3 response of 1321Nl astrocytoma cells to histamine stimulation and investigated the effects of PKC inhibition, downregulation and acute activation in order to establish whether a feedback loop was present and attempted to discern the isoform(s) involved and whether phospholipase D (PLD) contributes to PKC activation.1321N1 astrocytoma cells, passage 10-24, were maintained in DMEM supplemented with 25mM HEPES. 10% foetal calf serum, 2mM glutamine and antibiotics in 5% C02lhumidified air. Cultures were passaged weekly into 24 well multiplates or 80cm2 flasks. Cells were used 7-14 days after passage. lns(1,4,5)P3 mass was determined by radioreceptor assay using a bovine adrenal cortical binding protein preparation [Z]. Determination of PLD activity was made by measurement of the transphosphatidylation product, [33P]phosphatidylbutanol ([33P]PBut). in cells labelled for 24hrs with [33P]Pi and stimulated in the presence of SOmM butanol [3]. PKC isoforms were identified using Western blotting. Following separation by SDS PAGE proteins were transferred to nitrocellulose membranes which were probed with isoform specific antibodies. lmmunoreactive bands were visualised with ECL reagent and quantified by computer image analysis. Down-regulation experiments involved cells being treated in serum-free conditions with phorbol ester (lpM TPA) for 24hrs. Butanol (SOmM), PKC specific inhibitor ( 1 0~M Ro 31-8220). and acute phorbol ester treatment (100nM TPA) were all started lOmin prior to histamine(1 pM) stimulation.Assay of extracts of cells stimulated with a maximal concentration of histamine showed rapid, but small and variable, transient rises in Ins(l,4,5)P3 content, resting levels frequently being restored by 15s. When cells were stimulated in the presence of the PKC inhibitor Ro 31-8220 (1OpM) the magnitude and duration of the increase in Ins(l,4,5)P3 was greatly increased. Enhancement of the response was evident at the earliest time point, 5% (His: 4.2321.05, His+Ro: 10.14+0.59 pmoles/well, n=4, basal subtracted). increased to 15s (His: 4.04+1.61, His+Ro: 22.1251 2 4 pmoleslwell) and persisted at 30...