Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) is a minor component of the lipid bilayer but plays an important role in various cellular functions, including exocytosis and endocytosis. Recently, PI(4,5)P 2 was shown to form microdomains in the plasma membrane. In this study, we investigated the relationship between the spatial organization of PI(4,5)P 2 microdomains and exocytotic machineries in clonal rat pheochromocytoma PC12 cells. Both PI(4,5)P 2 and syntaxin, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein essential for exocytosis, exhibited punctate clusters in isolated plasma membranes. The number of PI(4,5)P 2 microdomains colocalizing with syntaxin clusters and large dense core vesicles (LDCVs) was decreased after catecholamine release. Alternatively, the expression of type I phosphatidylinositol-4-phosphate 5-kinase (PIP5KI) increased the number of PI(4,5)P 2 microdomains at syntaxin clusters with docked LDCVs and enhanced exocytotic activity, possibly by increasing the number of release sites. About half of the PI(4,5)P 2 microdomains were not colocalized with Thy-1, a specific marker of lipid rafts, and the colocalization of transfected PIP5KI with syntaxin clusters was observed. These results suggest that the formation of PI(4,5)P 2 microdomains at syntaxin clusters with docked LDCVs is essential for Ca 2؉ -dependent exocytosis.

The classical model of secretory vesicle recycling after exocytosis involves the retrieval of membrane (the omega figure) at a different site. An alternative model involves secretory vesicles transiently fusing with the plasma membrane (the 'kiss and run' mechanism) [1,2]. No continuous observation of the fate of a single secretory vesicle after exocytosis has been made to date. To study the dynamics of fusion immediately following exocytosis of insulin-containing vesicles, enhanced green fluorescent protein (EGFP) fused to the vesicle membrane protein phogrin [3] was delivered to the secretory vesicle membrane of INS-1 beta-cells using an adenoviral vector. The behaviour of the vesicle membrane during single exocytotic events was then examined using evanescent wave microscopy [4-6]. In unstimulated cells, secretory vesicles showed only slow Brownian movement. After a depolarizing pulse, most vesicles showed a small decrease in phogrin-EGFP fluorescence, and some moved laterally over the plasma membrane for approximately 1 microm. In contrast, secretory vesicles loaded with acridine orange all showed a transient (33-100 ms) increase in fluorescence intensity followed by rapid disappearance. Simultaneous observations of phogrin-EGFP and acridine orange indicated that the decrease in EGFP fluorescence occurred at the time of the acridine orange release, and that the lateral movement of EGFP-expressing vesicles occurred after this. Post-exocytotic retrieval of the vesicle membrane in INS-1 cells is thus slow, and can involve the movement of empty vesicles under the plasma membrane ('kiss and glide').

Calicheamicin-conjugated humanized anti-CD33 mouse monoclonal antibody, CMA-676, has recently been introduced to clinics as a promising drug to treat patients with acute myeloid leukemia (AML) in relapse. However, the mechanism of action of CMA-676 has not been well elucidated. The cytotoxic effect of CMA-676 on HL60, NOMO-1, NB4, NKM-1, K562, Daudi, and the multidrug-resistant sublines, NOMO-1/ADR and NB4/MDR, was investigated by cell cycle distribution and morphology. These studies were done by a video-microscopic system, DNA fragmentation, dye exclusion and 3H-thymidine uptake after analysis of CD33, CD34, P-glycoprotein (P-gp), multidrug resistance (MDR)-associated protein and lung-related protein on these cells. A dose-dependent, selective cytotoxic effect of CMA-676 was observed in cell lines that expressed CD33, and was dependent on the amount of CD33 and the proliferative speed of the cells. Sensitive cells were temporally arrested at the G2/M phase before undergoing morphological changes. CMA-676 is not effective on P-gp-expressing multidrug-resistant sublines compared with parental cell lines. MDR modifiers, MS209 and PSC833, restored the cytotoxic effect of CMA-676 in P-gp-expressing sublines. CMA-676 is a promising agent in the treatment of patients with AML that expresses CD33. The combined use of CMA-676 and MDR modifiers may increase the selective cytotoxic effect in multidrug-resistant AML.

Abstract. The growth cone is responsible for axonal growth, where membrane expansion is most likely to occur. Several recent reports have suggested that presynaptic proteins are involved in this process; however, the molecular mechanism details are unclear. We suggest that by cleaving a presynaptic protein syntaxin, which is essential in targeting synaptic vesicles as a target SNAP receptor (t-SNARE), neurotoxin C1 of Clostridium botulinum causes growth cone collapse and inhibits axonal growth. Video-enhanced microscopic studies showed (a) that neurotoxin C1 selectively blocked the activity of the central domain (the vesiclerich region) at the initial stage, but not the lamellipodia in the growth cone; and (b) that large vacuole formation occurred probably through the fusion of smaller vesicles from the central domain to the most distal segments of the neurite. The total surface area of the accumulated vacuoles could explain the membrane expansion of normal neurite growth. The gradual disappearance of the surface labeling by FITC-WGA on the normal growth cone, suggesting membrane addition, was inhibited by neurotoxin C1. The experiments using the peptides derived from syntaxin, essential for interaction with VAMP or a-SNAP, supported the results using neurotoxin C1. Our results demonstrate that syntaxin is involved in axonal growth and indicate that syntaxin may participate directly in the membrane expansion that occurs in the central domain of the growth cone, probably through association with VAMP and SNAPs, in a SNARE-like way.T hE nerve growth cone is a special structure at the leading edge of the extending axon and is responsible for axonal guidance and elongation. The growth cone consists of two distinct domains: the peripheral (p_)l and the central (C-) domains. P-domain, a microfilamentrich region including filopodia and lamellipodia, is responsible for motility (Dailey and Bridgman, 1993); C-domain, a vesicle-rich region (Forscher et al., 1987;Dailey and Bridgman, 1993), is the site where membrane expansion for axonal growth is suggested to occur (Goldberg and Address correspondence to Michihiro Igarashi, Department of Molecular and Cellular Neurobiology, Gunma University School of Medicine, Maebashi, Gunma 371, Portions of this work have appeared in abstract form (1995. Cell Struct. Funct. 20[Suppl.]:566a).1. Abbreviations used in this paper: C-domain, central domain; CNS, central nervous system; DPD, 2,2'-dipyridyl; DRG, dorsal root ganglion; P-domain, peripheral domain; SLO, streptolysin O; SNARE, SNAP receptor; VEC-DIC, video-enhanced contrast~lifferential interference contrast. Burmeister, 1986;Forscher et al., 1987;Pfenninger and Friedman, 1993). The growth cone alters its shape between active (advancing) and inactive (ceasing) forms in response to an appropriate (and inappropriate) stimulus for axonal growth (Kater and Mills, 1991;Schwab et al., 1993). An active growth cone is large with several filopodia, while an inactive one, known as a collapsed growth cone, is very small with few or no filo...

Total internal reflection fluorescence microscopy was used to detect single fluorescently labeled voltage-gated Shaker K ؉ channels in the plasma membrane of living cells. Tetramethylrhodamine (TMR) attached to specific amino acid positions in the voltagesensing S4 segment changed fluorescence intensity in response to the voltage-driven protein motions of the channel. The voltage dependence of the fluorescence of single TMRs was similar to that seen in macroscopic epi-illumination microscopy, but the exclusion of nonchannel fluorescence revealed that the dimming of TMR upon voltage sensor rearrangement was much larger than previously thought, and is due to an extreme, Ϸ20-fold suppression of the elementary fluorescence. The total internal reflection voltageclamp method reveals protein motions that do not directly open or close the ion channel and which have therefore not been detected before at the single-channel level. The method should be applicable to a wide assortment of membrane-associated proteins and should make it possible to relate the structural rearrangements of single proteins to simultaneously measured physiological cellsignaling events. D ynamic readouts of the structural rearrangements in membrane proteins have been obtained from changes in the fluorescence of site-specifically attached dyes (1-11). In ion channels the fractional fluorescent change (⌬F) can be large and very specific, differing in direction and amplitude and in which functional step is detected as the dye-attachment site is moved from one residue to the next (2, 9, 10). The mechanism of the fluorescence report is not known.The fluorescence studies have so far been confined to large ensembles of proteins, where the variation from protein to protein in occupancy of distinct states blurs the transitions between them, even when activating signals are synchronized by voltage clamping. Such blurring can be avoided in singlemolecule determinations (12)(13)(14). Obstacles to optical detection have so far limited single-molecule optical studies of protein rearrangement to purified preparations, out of the native cellular context. We have overcome these obstacles for the optical detection of structural rearrangements in single-membrane proteins in living cells, enabling functional transitions that do not open or close gates to be detected on the single-channel level. Materials and Methods Molecular Biology.Fluorescence experiments were performed on nonconducting (W434F) (15), ball-deleted (⌬6-46) (16) ShH4 Shaker channels (17), after the removal of two native cysteines (C245V and C462A) (2) to ensure that membrane-impermeant fluorescent thiols would attach exclusively to a known position of cysteine addition. Site-directed mutagenesis, cRNA synthesis, and cRNA injection into Xenopus oocytes were as described (3), leading to high-density channel expression in all of the experiments.Oocyte Preparation. The vitelline membrane was found to be about 3 m thick by scanning electron microscopy (not shown) and to refract or scatter the excitation li...