Membranes vesicular

Human KB carcinoma cells resistant to vinblastine and other drugs have been shown to exhibit increased membrane vesicular binding of tritiated vinblastine, and this binding is correlated with photoaffinity labeling of a 150,000- to 170,000-dalton protein in the vesicles. Labeling of this protein is inhibited by vinblastine, vincristine, and verapamil but not by colchicine (79). The failure of colchicine to inhibit the labeling of the membrane protein is unexpected since the cells from which the protein was isolated are resistant to colchicine as well as vinblastine. 

Fig. 8 Phase and fluorescence micrographs of membranous vesicular structures formed from a Murchison meteorite extract (left) compared to vesicles formed by a 20 mM de-canoic acid-decanol mixture [72] (center) and a vesicular structure produced by the photoproduct of an interstellar-ice analog [31]. The vesicles produced by the photochemical ice analog product were allowed to capture pyranine, a fluorescent anionic dye, to demonstrate that a true membrane was present. Scale bars show 20, 10, and 5 pm, from left to right...

Cell membranes are two-dimensional fluids that exhibit a wide range of dynamic behaviors. Recent technical advances have enabled unprecedented views of membrane dynamics in living cells. In this technical review, we provide a brief overview of three well-studied examples of membrane dynamics lateral diffusion of proteins and lipids in the plane of the membrane, vesicular trafficking between intracellular compartments, and exchange of proteins on and off membranes. We then discuss experimental approaches to monitor membrane protein and lipid dynamics, and we place a special emphasis on the use of genetically encoded fluorescent probes and live cell-imaging techniques. 

Membrane-Based Assays Membranes prepared from cells expressing transporters have been widely used to study the function of ABC efflux pumps and to identify their substrates or inhibitors. Currently, there are two major membrane-based assays the ATPase assay and the membrane vesicular transport (uptake) assay. Compared to the cell-based assay, the membrane-based assay has several advantages including (1) the assay can be used to characterize the effect of a xenobiotic on one specific efflux transporter (2) the assay can be easily employed in a high throughput mode (3) membranes are easy to be maintained after preparation and (4) the assay is easy to conduct. 

Newport, J., and Spann, T. (1987). Disassembly of the nucleus in mitotic extracts Membrane vesicular-ization, lamin disassembly and chromatin condensation are independent processes. Cell (Cambridge, Mass.) 482, 219-230. 

A typical biomembrane consists largely of amphiphilic lipids with small hydrophilic head groups and long hydrophobic fatty acid tails. These amphiphiles are insoluble in water (<10 ° mol L ) and capable of self-organization into uitrathin bilaycr lipid membranes (BLMs). Until 1977 only natural lipids, in particular phospholipids like lecithins, were believed to form spherical and related vesicular membrane structures. Intricate interactions of the head groups were supposed to be necessary for the self-organization of several ten thousands of... 

The pharmacology of amphetamine is considerably more complex. It does not only block monoamine reuptake, but also directly inhibits the vesicular monoamine transporter, causing an increase in cytosolic but not vesicular dopamine concentration. This may lead to reverse transport of the amines via the membrane-bound transporters. Further mechanisms of amphetamine action are direct MAO inhibition and indirect release of both dopamine and serotonin in the striatum. 

Sialin was first identified as the product of the gene defective in sialidosis, a lysosomal storage disorder. The transporter mediates the movement of sialic acid out of lysosomes by coupling to the proton electrochemical gradient across the lysosomal membrane. Unlike the vesicular neurotransmitter transporters which are antiporters, sialin is a sympoiter with sialic acid and protons both moving out of the lysosome. 

The exocytotic release of neurotransmitters from synaptic vesicles underlies most information processing by the brain. Since classical neurotransmitters including monoamines, acetylcholine, GABA, and glutamate are synthesized in the cytoplasm, a mechanism is required for their accumulation in synaptic vesicles. Vesicular transporters are multitransmembrane domain proteins that mediate this process by coupling the movement of neurotransmitters to the proton electrochemical gradient across the vesicle membrane. 

Synaptic vesicles isolated from brain exhibit four distinct vesicular neurotransmitter transport activities one for monoamines, a second for acetylcholine, a third for the inhibitory neurotransmitters GABA and glycine, and a fourth for glutamate [1], Unlike Na+-dependent plasma membrane transporters, the vesicular activities couple to a proton electrochemical gradient (A. lh+) across the vesicle membrane generated by the vacuolar H+-ATPase ( vacuolar type proton translocating ATPase). Although all of the vesicular transport systems rely on ApH+, the relative dependence on the chemical and electrical components varies (Fig. 1). The... 

The vesicular monoamine transporters (VMATs) were identified in a screen for genes that confer resistance to the parkinsonian neurotoxin MPP+ [2]. The resistance apparently results from sequestration of the toxin inside vesicles, away from its primary site of action in mitochondria. In addition to recognizing MPP+, the transporter s mediate the uptake of dopamine, ser otonin, epinephrine, and norepinephrine by neurons and endocrine cells. Structurally, the VMATs show no relationship to plasma membrane monoamine transporters. 

VMATs are not inhibited by drugs such as cocaine, tricyclic antidqnessants and selective serotonin reuptake inhibitors that affect plasma membrane monoamine transport. Amphetamines have relatively selective effects on monoaminergic cells due to selective uptake by plasma membrane monoamine transporters, but their effect appears to be mediated by their ability as weak bases to reduce ApH, the driving force for vesicular monoamine transport that leads to efflux of the vesicular contents into the cytoplasm. 

Several compounds that inhibit vesicular glutamate transport have been identified These include the dyes Evans Blue and Rose Bengal. In addition, the stilbene derivative 4,4 -diisothiocyanatostilbene-2,2 -disulfonic acid (DEDS), a compound commonly used as a specific inhibitor of anion channels, inhibits vesicular glutamate transport. Most known inhibitors have limited use as they are membrane impermeant, with the exception of Rose Bengal. 

In the first step, lipid model membranes have been generated (Fig. 15) on the air/liquid interface, on a glass micropipette (see Section VIII.A.1), and on an aperture that separates two cells filled with subphase (see Section VIII.A.2). Further, amphiphilic lipid molecules have been self-assembled in an aqueous medium surrounding unilamellar vesicles (see Section VIII.A.3). Subsequently, the S-layer protein of B. coagulans E38/vl, B. stearother-mophilus PV72/p2, or B. sphaericus CCM 2177 have been injected into the aqueous subphase (Fig. 15). As on solid supports, crystal growth of S-layer lattices on planar or vesicular lipid films is initiated simultaneously at many randomly distributed nucleation... 

Certain other non-KDEL-containing proteins destined for the membranes of the ER also pass to the Golgi and then return, by retrograde vesicular transport, to the ER to be inserted therein (see below). 

Based largely on a proposal by Rothman and colleagues, anterograde vesicular transport can be considered to occur in eight steps (Figure 46-7). The basic concept is that each transport vesicle bears a unique address marker consisting of one or more v-SNARE proteins, while each target membrane bears one or more complementary t-SNARE proteins with which the former interact specifically. 

Fig. 1.—Diagrammatic Representation of the Three Steps in the Taste-cell Transduction. Step 1, interaction of stimulus (S) with membrane-bound receptor (R) to form stimulus-receptor complex (SR) step 2, conformational change (SR) to (SR), brought about by interaction of S with R (this change initiates a change in plasma-membrane conformation of taste cells, probably below the level of the tight junction) and step 3, conformational changes of the membrane result in lowered membrane resistance, and the consequential influx on intracellular ionic species, probably Na. This influx generates the receptor potential which induces synaptic vesicular release to the innervating, sensory nerve, leading to the generator potential.

Synapsins comprise a family of phosphoproteins that are found only in association with SSVs. Although they account for only about 9% of the total vesicular membrane protein they probably cover a large proportion of their surface. So far, synapsins la, Ib, Ila, Ilb and III, which are the products of different genes, have been identified. 

Figure 4.11 Dephosphorylated synapsin, associated with SSVs, is thought to form a heteromeric complex with CAM kinase II (also partially embedded in the vesicular membrane) and actin filaments. An increase in intracellular Ca + triggers phosphorylation of S3mapsin I which dissociates from the vesicular membrane. This frees the vesicles from the fibrin microfilaments and makes them available for transmitter release at the active zone of the nerve terminal...

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