Continuous and transient vesicle cycling at a ribbon synapse

Optical methods were used to study the Ca2+ dependence of vesicle cycling in bipolar cells isolated from goldfish retinas. Uniformly raising the Ca2+ concentration to between 0.8 and 20 microM produced a continuous vesicle cycle of balanced exocytosis and endocytosis with a maximum rate equivalent to the turnover of the entire surface membrane of a terminal every 2 min (or approximately 900 vesicles sec-1). Increasing the Ca2+ concentration above 20 microM inhibited continuous vesicle cycling. In contrast, influx of Ca2+ through voltage-gated channels produced a transient burst of exocytosis that increased the surface area of a terminal by a maximum of 12% (equivalent to the addition of 13,000 vesicles). Endocytosis was delayed until after Ca2+ influx stopped and the average Ca2+ concentration in the terminal declined. Hence, a single terminal has mechanisms for both continuous and transient vesicle cycling.     

Biochemical and functional heterogeneity of rat cerebrum microsomal membranes in relation to SERCA Ca(2+)-ATPases and Ca2+ release channels

Rat cerebrum microsomes were subfractionated on isopycnic linear sucrose (20-42%) density gradients. The Ca2+ loading/release properties and the distribution of intracellular Ca2+ store channels, inositol 1,4,5-trisphosphate (IP3) receptor and ryanodine (Ry) receptor, and SERCA pumps, were monitored in each subfraction by ligand binding and 45Ca2+ loading/release assays. Three different classes of vesicles were identified: (i) heavy density vesicles with high content of Ry receptors and Ca2+ pumps and high thapsigargin (TG)-sensitivity of Ca2+ loading; (ii) intermediate sucrose density vesicles with high content of IP3 receptor, high IP(S)3-sensitivity of Ca2+ loading and low content of Ry receptors; and (iii) light sucrose density vesicles with high content of Ry receptors, low content of IP3 receptors and low content of SERCA pumps highly sensitive to TG. Isolation of molecularly heterogeneous rat cerebrum microsomes and identification of specific Ca2+ loading/release properties support the presence of multiple, potentially active, heterogeneous rapidly exchanging Ca2+ stores in rat cerebrum.     

Subchronic toxicity of PCB 105 (2,3,3'',4,4''-pentachlorobiphenyl) in rats

Increases in the intracellular free calcium concentration are of great importance to the initiation of development in deuterostomes. Their involvement has not yet been clearly defined in protostomes. We used endogenous ligands (IP3, cADPR, ryanodine and NAADP) and pharmacological agents (thapsigargin [Tg], thimerosal, caffeine and heparin) to study smooth endoplasmic reticulum Ca2+ pump and release mechanisms in eggs of an annelid, Chaetopterus. Oocyte homogenates effectively sequestered Ca2+ and released it in response to IP3 in a concentration-dependent manner. Repeated additions of IP3 were unable to cause further release. Heparin inhibited Ca2+ release in response to IP3. The homogenates also released Ca2+ in response to thimerosal, and this release was sensitive to heparin. Two antibodies to IP3 receptors recognized an appropriate band in Chaetopterus egg lysates. These results indicate that the oocytes possess type-1 IP3-gated Ca2+ channels. Neither calcium itself, nor strontium, cADPR, ryanodine, caffeine nor NAADP released appreciable Ca2+. At low concentrations, Tg caused a slow release of Ca2+; at higher concentrations, it elicited a rapid release. Release of Ca2+ by Tg activated development. Since one theory of fertilization invokes the introduction of a Ca2+ releasing soluble protein into the egg upon sperm-egg fusion, we also tested whether soluble extracts of Chaetopterus sperm could stimulate Ca2+ release in Chaetopterus egg homogenates. There was no Ca2+ release when the sperm extract was added to the homogenate; however, homogenates exposed to sperm extract became refractory to IP3. Thus, Ca2+ release at fertilization in these oocytes occurs through IP3-gated channels.     

Bradykinin inhibits M current via phospholipase C and Ca2+ release from IP3-sensitive Ca2+ stores in rat sympathetic neurons

A variety of intracellular signaling pathways can modulate the properties of voltage-gated ion channels. Some of them are well characterized. However, the diffusible second messenger mediating suppression of M current via G protein-coupled receptors has not been identified. In superior cervical ganglion neurons, we find that the signaling pathways underlying M current inhibition by B2 bradykinin and M1 muscarinic receptors respond very differently to inhibitors. The bradykinin pathway was suppressed by the phospholipase C inhibitor U-73122, by blocking the IP3 receptor with pentosan polysulfate or heparin, and by buffering intracellular calcium, and it was occluded by allowing IP3 to diffuse into the cytoplasm via a patch pipette. By contrast, the muscarinic pathway was not disrupted by any of these treatments. The addition of bradykinin was accompanied by a [Ca2+]i rise with a similar onset and time to peak as the inhibition of M current. The M current inhibition and the rise of [Ca2+]i were blocked by depletion of Ca2+ internal stores by thapsigargin. We conclude that bradykinin receptors inhibit M current of sympathetic neurons by activating phospholipase C and releasing Ca2+ from IP3-sensitive Ca2+ stores, whereas muscarinic receptors do not use the phospholipase C pathway to inhibit M current channels.     

Submillisecond kinetics of glutamate release from a sensory synapse

Exocytosis-mediated glutamate release from ribbon-type synaptic terminals of retinal bipolar cells was studied using AMPA receptors and simultaneous membrane capacitance measurements. Release onset (delay <0.8 ms) and offset were closely tied to Ca2+ channel opening and closing. Asynchronous release was not copious and we estimate that there are approximately 5 Ca2+ channels per docked synaptic vesicle. Depending on Ca2+ current amplitude, release occurred in a single fast bout or in two successive bouts with fast and slow onset kinetics. The second, slower bout may reflect a mobilization rate of reserve vesicles toward fusion sites that is accelerated by increasing Ca2+ influx. Bipolar cell synaptic ribbons thus are remarkably versatile signal transducers, capable of transmitting rapidly changing sensory input, as well as sustained stimuli, due to their large pool of releasable vesicles.     

Concentration-dependent modulations of potassium and calcium currents of rat osteoblastic cells by arachidonic acid

We show that the voltage-gated K+ and Ca2+ currents of rat osteoblastic cells are strongly modulated by arachidonic acid (AA), and that these modulations are very sensitive to the AA concentration. At 2 or 3 microM, AA reduces the amplitude and accelerates the inactivation of the K+ current activated by depolarization; at higher concentrations (> or = 5 microM), AA still blocks this K+ current, but also induces a very large noninactivating K+ current. At 2 or 3 microM, AA enhances the T-type Ca2+ current, close to its threshold of activation, whereas at 10 microM, it blocks that current. AA (1-10 microM) also blocks the dihydropyridine-sensitive L-type Ca2+ current. Thus, the effect of AA on Ca2+ entry through voltage-gated Ca2+ channels can change qualitatively with the AA concentration: at 2 or 3 microM, AA will favor Ca2+ entry through T channels, both by lowering the voltage-gated K+ conductance and by increasing the T current, whereas at 10 microM, AA will prevent Ca2+ entry through voltage-gated Ca2+ channels, both by inducing a K+ conductance and by blocking Ca2+ channels.     

The germinal vesicle of the mouse oocyte contains elements of the phosphoinositide cycle: what is their role at meiosis resumption?

The role of the nuclear phosphoinositide (PI) cycle during meiotic resumption in mouse oocytes was examined. First, using indirect immunofluorescence staining with specific monoclonal antibodies (mAbs) against elements of this cycle, the presence of inositol trisphosphate receptors (IP3Rs) (IP3R-1 or IP3R-3) or phosphoinositide-phospholipase (PLC) isoforms (PLC beta 1 or PLC gamma 1) was monitored in the germinal vesicle (GV). Using confocal laser scanning microscopy, we analysed the effects of the nuclear microinjection of these antibodies on both spontaneous nuclear calcium oscillations and meiosis resumption. Immunostainings showed that IP3R-1 and PLC beta 1 isoforms were both present in the GV, whereas IP3R-3 and PLC gamma 1 isoforms were not. The anti-IP3R-1 mAbs or the anti-PLC beta 1 mAbs microinjected into the GV, induced inhibition of both the nuclear Ca2+ oscillations and the meiotic process, whereas the anti-IP3R-3 mAbs and the anti-PLC gamma 1 mAbs did not. We concluded that a specific nuclear PI cycle is present in the mouse oocyte and meiosis resumption requires a specific nuclear phosphoinositide-dependent Ca2+ signal.     

Intracellular calcium release channel expression during embryogenesis

The release of intracellular calcium (Ca2+) via either inositol 1,4, 5-trisphosphate receptors (IP3R) or ryanodine receptors (RyR) activates a wide variety of signaling pathways in virtually every type of cell. In the present study we demonstrate that at early stages of development IP3R mRNA and functional IP3-gated Ca2+ release channels are widely expressed in virtually all tissues in murine embryos. As organogenesis proceeds, more specialized RyR channels are expressed in many cell types and the triggering mechanisms for intracellular Ca2+ release become more diverse to include IP3-dependent and voltage-dependent and Ca2+-induced Ca2+ release. As development proceeds virtually all cell types continue to express IP3R channels but in excitable cells including skeletal and cardiac muscles the major Ca2+ release channels are RyRs. This developmental switch from predominantly IP3-mediated to both IP3-mediated and IP3-independent pathways for intracellular Ca2+ release is consistent with data showing that IP3R plays an important regulatory role in cellular proliferation and apoptosis, whereas RyR is required for other cellular functions including muscle contraction.     

Mechanisms determining the time course of secretion in neuroendocrine cells

Transmitter release from chromaffin cells differs from that in synapses in that it persists for a longer time after Ca2+ entry has stopped. This prolonged secretion is not due to a delay between vesicle fusion and transmitter release, nor to slow detection of released substance: step increases in capacitance due to single vesicle fusion precede the release detected by amperometry by only a few milliseconds. The persistence of secretion after a depolarization is reduced by addition of mobile calcium buffer. This suggests that most of the delay is due to diffusion of Ca2+ between channels and release sites, implying that Ca2+ channels and secretory vesicles are not colocalized in chromaffin cells, in contrast to presynaptic active zones.     

Inositol trisphosphate and cyclic ADP ribose as long range messengers generating local subcellular calcium signals

The process of messenger-mediated release of Ca2+ from intracellular stores, which is of great importance in virtually all cell types including neurons, can best be studied in cells lacking voltage-gated Ca2+ channels in the plasma membrane. In pancreatic acinar cells agonist-evoked repetitive cytosolic Ca2+ spikes are due to release of Ca2+ via inositoltrisphosphate (IP3) and ryanodine receptors and reuptake into the stores via thapsigargin-sensitive Ca2+ pumps. At low acetylcholine (ACh) or cholecystokinin concentrations the cytosolic Ca2+ spikes are mostly confined to the secretory granule area of the polarized pancreatic acinar cells. Similar results can be obtained by intracellular infusion of IP3 (or one of its non-metabolizable analogues) or cyclic ADP ribose. This suggests that high affinity IP3 and ryanodine receptors are concentrated in the secretory granule area. We have generated an 'artificial synapse' on isolated acinar cells by having a cell-attached patch pipette filled with ACh on the basal membrane. Initially, ACh is prevented from making contact with the receptors by the negative potential applied to the pipette. When the pipette polarity is switched to positive ACh can bind to its receptors. Using digital Ca2+ imaging it could be seen that the first cytosolic rise often occurred in the secretory granule area, a considerable distance away from the site of the agonist-receptor interaction. This shows the long-range action of the messenger(s) IP3 and or cyclic ADP ribose generated by the ACh-receptor interaction. The local Ca2+ spikes in the secretory granule area are sufficient for exocytotic secretory responses as seen in capacitance measurements.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of polyelectrolyte complex (PEC) on human periodontal ligament fibroblast (HPLF) function. I. Three-dimensional structure of HPLF cultured on PEC

The functional effect of activating Ca2+-permeable neuronal nicotinic acetylcholine receptors (nAChRs) on vesicle secretion was studied in PC12 cells. Single cells were patch-clamped in the whole-cell configuration and stimulated with either brief pulses of nicotine to activate the Ca2+-permeable nAChRs or with voltage steps to activate voltage-dependent Ca2+ channels. Membrane capacitance was used as a measure of vesicle secretion. Activation of nAChRs by nicotine application to cells voltage clamped at -80 mV evoked secretion. This secretion was completely abolished by nicotinic antagonists. When the cells were voltage clamped at +20 mV in the presence of Cd2+ to block voltage-activated Ca2+ channels, nicotine elicited a small amount of secretion. Most interestingly, when the nAChRs were activated coincidentally with voltage-dependent Ca2+ channels, secretion was augmented approximately twofold over the secretion elicited with voltage-dependent Ca2+ channels alone. Our data suggest that Ca2+ influx via nAChRs affects Ca2+-dependent cellular functions, including vesicle secretion. In addition to the secretion evoked by nAChR activation at hyperpolarized potentials, we demonstrate that even at depolarized potentials, nAChRs provide an important Ca2+ entry pathway underlying Ca2+-dependent cellular processes such as exocytosis.     

Are exocytosis mechanisms neurotransmitter specific?

Neurotransmission is a multistage regulated process in which a variety of active molecules contained in vesicles are liberated in response to specific stimuli from different types of neurone or related cells. This includes the release of fast neurotransmitters such as amino acids and acetylcholine from central and peripheral synapses, but also that of relatively slow-acting polypeptides from central and peripheral neurones or neuroendocrine cells. Considerable progress has been made over recent years in the understanding at a molecular level of the mechanism of regulated exocytosis, a crucial phase in this phenomenon. The currently proposed overall mechanism, which incorporates the "SNARE" hypothesis for vesicle-membrane docking and fusion, is based on data from experimental models ranging from brain synaptosomes to mast cells. Since the kinetics of the models studied and the physiological effects of the neurotransmitters implicated vary so much, it is pertinent to question whether a general mechanism can be proposed from such experimental data. This review examines known differences in putative exocytotic mechanisms for the various systems studied and attempts to relate these to the nature of the active substances released. Differences exist in each step of the exocytosis process and include the channel through which Ca2+ enters to trigger it or the internal Ca2- source, the type of vesicle in which the transmitter is packaged, the way vesicles are translocated to the surface membrane or how they dock and fuse with it. Major differences have been reported in release mechanisms of different types of vesicle, but minor differences also exist within the same vesicle class. Thus small synaptic vesicles and large dense core vesicles are translocated by distinct processes and the Ca2+ channels, Ca2+ sensors and docking proteins involved in other steps are not identical in all neuronal phenotypes. It may be concluded that each of these differences has evolved to accommodate the different physiological requirements of the neuromodulator released.     

Regulation of cyclooxygenase-2 pathway by HER2 receptor

In the present work, we characterized the receptor properties and the conductive features of the inositol (1,4,5)-trisphosphate (IP3)-activated Ca2+ channels present in excised plasma-membrane patches obtained from mouse macrophages and A431 cells. We found that the receptor properties of the channels tested were similar to those of the IP3 receptor (IP3R) expressed in the endoplasmic reticulum (ER) membrane. These properties include activation by IP3, inhibition by heparin, time-dependent inactivation by high IP3 concentrations, activation by guanosine 5'o-thiotriphosphate and regulation by arachidonic acid. On the other hand, in terms of conductive properties, the channel closely resembles Ca2+-release-activated Ca2+ channels (Icrac). These conductive properties include extremely low conductance (approximately 1 pS), very high selectivity for Ca2+ over K+ (PCa/PK>1000), inactivation by high intracellular Ca2+ concentration and, importantly, strong inward rectification. Notably, the same channel was activated by: (1) agonists in the cell-attached mode of channel recording, and (2) cytosolic IP3 after patch excision. Although the possibility cannot be completely excluded that a novel type of IP3R is expressed exclusively in the plasma membrane, in their entirety our findings suggest that the plasma membrane of mouse macrophages and A431 cells contains Icrac-like Ca2+ channels coupled to an IP3-responsive protein which displays properties similar to those of the IP3R expressed in the ER membrane.     

Formation and turnover of NSF- and SNAP-containing "fusion" complexes occur on undocked, clathrin-coated vesicle-derived membranes

Specificity of vesicular transport is determined by pair-wise interaction between receptors (SNAP receptors or SNAREs) associated with a transport vesicle and its target membrane. Two additional factors, N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment protein (SNAP) are ubiquitous components of vesicular transport pathways. However, the precise role they play is not known. On the basis that NSF and SNAP can be recruited to preformed SNARE complexes, it has been proposed that NSF- and SNAP-containing complexes are formed after SNARE-dependent docking of transport vesicles. This would enable ATPase-dependent complex disassembly to be coupled directly to membrane fusion. Alternatively, binding and release of NSF/SNAP may occur before vesicle docking, and perhaps be involved in the activation of SNAREs. To gain more information about the point at which so-called 20S complexes form during the transport vesicle cycle, we have examined NSF/SNAP/SNARE complex turnover on clathrin-coated vesicle-derived membranes in situ. This has been achieved under conditions in which the extent of membrane docking can be precisely monitored. We demonstrate by UV-dependent cross-linking experiments, coupled to laser light-scattering analysis of membranes, that complexes containing NSF, SNAP, and SNAREs will form and dissociate on the surface of undocked transport vesicles.     

Dantrolene inhibition of sarcoplasmic reticulum Ca2+ release by direct and specific action at skeletal muscle ryanodine receptors

The skeletal muscle relaxant dantrolene inhibits the release of Ca2+ from the sarcoplasmic reticulum during excitation-contraction coupling and suppresses the uncontrolled Ca2+ release that underlies the skeletal muscle pharmacogenetic disorder malignant hyperthermia; however, the molecular mechanism by which dantrolene selectively affects skeletal muscle Ca2+ regulation remains to be defined. Here we provide evidence of a high-affinity, monophasic inhibition by dantrolene of ryanodine receptor Ca2+ channel function in isolated sarcoplasmic reticulum vesicles prepared from malignant hyperthermia-susceptible and normal pig skeletal muscle. In media simulating resting myoplasm, dantrolene increased the half-time for 45Ca2+ release from both malignant hyperthermia and normal vesicles approximately 3.5-fold and inhibited sarcoplasmic reticulum vesicle [3H]ryanodine binding (Ki approximately 150 nM for both malignant hyperthermia and normal). Inhibition of vesicle [3H]ryanodine binding by dantrolene was associated with a decrease in the extent of activation by both calmodulin and Ca2+. Dantrolene also inhibited [3H]ryanodine binding to purified skeletal muscle ryanodine receptor protein reconstituted into liposomes. In contrast, cardiac sarcoplasmic reticulum vesicle 45Ca2+ release and [3H]ryanodine binding were unaffected by dantrolene. Together, these results demonstrate selective effects of dantrolene on skeletal muscle ryanodine receptors that are consistent with the actions of dantrolene in vivo and suggest a mechanism of action in which dantrolene may act directly at the skeletal muscle ryanodine receptor complex to limit its activation by calmodulin and Ca2+. The potential implications of these results for understanding how dantrolene and malignant hyperthermia mutations may affect the voltage-dependent activation of Ca2+ release in intact skeletal muscle are discussed.     

Protein kinase A-activated chloride channel is inhibited by the Ca(2+)-calmodulin complex in cardiac sarcoplasmic reticulum

Cardiac sarcoplasmic reticulum (SR) has several chloride (Cl-) channels, which may neutralize the charge across the SR membrane generated by Ca2+ movement. We recently reported a novel 116-picosiemen Cl- channel that is activated by protein kinase A-dependent phosphorylation in cardiac SR. This Cl- channel may serve as a target protein in the receptor-dependent regulation of cardiac excitation-contraction coupling. To understand further regulatory mechanisms, the effects of Ca2+ on the Cl- channel were studied using the planar lipid bilayer-vesicle fusion technique. In the presence of calmodulin (CaM, 0.1 mumol/L per microgram SR vesicles), Ca2+ (3 mumol/L to 1 mmol/L) added to the cis solution reduced the channel openings in a concentration-dependent fashion, whereas Ca2+ (1 nmol/L to 1 mmol/L) alone or CaM (0.1 to 1 mumol/L per microgram SR vesicles) with 1 nmol/L Ca2+ did not affect the channel activity. This inhibitory effect of Ca2+ in the presence of CaM was prevented by CaM inhibitors N-(6 aminohexyl)-5-chloro-1-naphthalenesulfonamide and calmidazolium but not by CaM kinase II inhibitor KN62. These results suggest that the Ca(2+)-CaM complex itself, but not CaM kinase II, is involved in this channel inhibition. Thus, the cardiac SR 116-picosiemen Cl- channel is regulated not only by protein kinase A-dependent phosphorylation but also by the cytosolic Ca(2+)-CaM complex. This is a novel second messenger-mediated regulation of Cl- channels in cardiac SR membrane.     

Effect of dry fibrin sealant dressings versus gauze packing on blood loss in grade V liver injuries in resuscitated swine

In mammalian fertilization, inositol 1,4,5-trisphosphate receptor (IP3R)-dependent Ca2+ release is a crucial signaling event that originates from the vicinity of sperm-egg interaction and spreads as a wave throughout the egg cytoplasm. While it is known that Ca2+ is released by the type 1 IP3R in the egg cortex, the potential involvement of other isoform types responsible for the Ca2+ rise in the mouse egg (interior) and their spatial distribution are not known. In addition, the biochemical basis has not been definitively established for the development of increased sensitivity to inositol 1,4,5-trisphosphate (IP3) during meiotic maturation. Using specific antibodies to the type 1, 2, and 3 IP3R, we tested the hypotheses that different IP3R isoforms are responsible for the internal Ca2+ elevation and that they contribute to the maturation-associated acquisition of IP3 sensitivity. In both preovulatory oocytes and ovulated eggs of CF-1 mice, immunofluorescence revealed that types 1 and 2 isoforms were present in the cell cortex and interior. Type 1 was observed throughout the cytoplasm, and Western analysis indicated a 1.9-fold maturation-associated increase. In contrast, the signals detected for the type 2 (high-affinity) isoform and type 3 were present to a lesser extent, with type 2 restricted to isolated islands (similar to aggregates of vesicles detected by electron microscopy), which, in the cortex, may amplify early sperm-egg signaling events. The cortical-to-perinuclear localization of the receptor and cortical vesicle aggregates imply an efficient mechanism for propagating Ca2+ release from the cortex into the interior of the egg to activate development, and the isoform localization analysis indicates a clear spatial and biochemical heterogeneity. Types 1 and 2 isoforms were also present in granulosa cells.     

Activation of nicotinic receptor-induced postsynaptic responses to luteinizing hormone-releasing hormone in bullfrog sympathetic ganglia via a Na+-dependent mechanism

Nicotine at very low doses (5-30 nM) induced large amounts of luteinizing hormone-releasing hormone (LHRH) release, which was monitored as slow membrane depolarizations in the ganglionic neurons of bullfrog sympathetic ganglia. A nicotinic antagonist, d-tubocurarine chloride, completely and reversibly blocked the nicotine-induced LHRH release, but it did not block the nerve-firing-evoked LHRH release. Thus, nicotine activated nicotinic acetylcholine receptors and produced LHRH release via a mechanism that is different from the mechanism for evoked release. Moreover, this release was not caused by Ca2+ influx through either the nicotinic receptors or the voltage-gated Ca2+ channels because the release was increased moderately when the extracellular solution was changed into a Ca2+-free solution that also contained Mg2+ (4 mM) and Cd2+ (200 microM). The release did not depend on Ca2+ release from the intraterminal Ca2+ stores either because fura-2 fluorimetry showed extremely low Ca2+ elevation (approximately 30 nM) in response to nicotine (30 nM). Moreover, nicotine evoked LHRH release when [Ca2+] elevation in the terminals was prevented by loading the terminals with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and fura-2. Instead, the nicotine-induced release required extracellular Na+ because substitution of extracellular NaCl with N-methyl-D-glucamine chloride completely blocked the release. The Na+-dependent mechanism was not via Na+ influx through the voltage-gated Na+ channels because the release was not affected by tetrodotoxin (1-50 microM) plus Cd2+ (200 microM). Thus, nicotine at very low concentrations induced LHRH release via a Na+-dependent, Ca2+-independent mechanism.     

Temporal differences in the appearance of NEP-B78 and an LBR-like protein during Xenopus nuclear envelope reassembly reflect the ordered recruitment of functionally discrete vesicle types

In this work, we have used novel mAbs against two proteins of the endoplasmic reticulum and outer nuclear membrane, termed NEP-B78 and p65, in addition to a polyclonal antibody against the inner nuclear membrane protein LBR (lamin B receptor), to study the order and dynamics of NE reassembly in the Xenopus cell-free system. Using these reagents, we demonstrate differences in the timing of recruitment of their cognate membrane proteins to the surface of decondensing chromatin in both the cell-free system and XLK-2 cells. We show unequivocally that, in the cell-free system, two functionally and biochemically distinct vesicle types are necessary for NE assembly. We find that the process of distinct vesicle recruitment to chromatin is an ordered one and that NEP-B78 defines a vesicle population involved in the earliest events of reassembly in this system. Finally, we present evidence that NEP-B78 may be required for the targeting of these vesicles to the surface of decondensing chromatin in this system. The results have important implications for the understanding of the mechanisms of nuclear envelope disassembly and reassembly during mitosis and for the development of systems to identify novel molecules that control these processes.