Correlative light-electron microscopy reveals the tubular-saccular ultrastructure of carriers operating between Golgi apparatus and plasma membrane.

Transport intermediates (TIs) have a central role in intracellular traffic, and much effort has been directed towards defining their molecular organization. Unfortunately, major uncertainties remain regarding their true structure in living cells. To address this question, we have developed an approach based on the combination of the green fluorescent protein technology and correlative light-electron microscopy, by which it is possible to monitor an individual carrier in vivo and then take a picture of its ultrastructure at any moment of its life-cycle. We have applied this technique to define the structure of TIs operating from the Golgi apparatus to the plasma membrane, whose in vivo dynamics have been characterized recently by light microscopy. We find that these carriers are large (ranging from 0.3-1.7 microm in maximum diameter, nearly half the size of a Golgi cisterna), comprise almost exclusively tubular-saccular structures, and fuse directly with the plasma membrane, sometimes minutes after docking to the fusion site.

CtBP/BARS induces fission of Golgi membranes by acylating lysophosphatidic acid.

Membrane fission is essential in intracellular transport. Acyl-coenzyme As (acyl-CoAs) are important in lipid remodelling and are required for fission of COPI-coated vesicles. Here we show that CtBP/BARS, a protein that functions in the dynamics of Golgi tubules, is an essential component of the fission machinery operating at Golgi tubular networks, including Golgi compartments involved in protein transport and sorting. CtBP/BARS-induced fission was preceded by the formation of constricted sites in Golgi tubules, whose extreme curvature is likely to involve local changes in the membrane lipid composition. We find that CtBP/BARS uses acyl-CoA to selectively catalyse the acylation of lysophosphatidic acid to phosphatidic acid both in pure lipidic systems and in Golgi membranes, and that this reaction is essential for fission. Our results indicate a key role for lipid metabolic pathways in membrane fission.

Coalescence of Golgi fragments in microtubule-deprived living cells.

The process of stack coalescence, an important mechanism of Golgi recovery from mitosis, was examined using novel experimental paradigms. In living cells with disrupted (by nocodazole) microtubules, galactosyl transferase-GFP-labelled Golgi fragments constantly appeared, grew, sometimes moved with a speed of 1-2 microns/min, coalesced or gradually diminished and disappeared. The rate of Golgi fragment turnover and coalescence was highly balanced to maintain a constant number of Golgi units per cell. Moreover some Golgi islands appear and some received new GalTase-GFP after photobleaching of cell cytoplasm. Short tubules extending from the rims of scattered Golgi fragments frequently formed bridges between ministacks, inducing their coalescence. The frequency of coalescence could also be inhibited by disruption of actin microfilaments. After the Golgi redistribution into endoplasmic reticulum induced by brefeldin A, either the growth of small Golgi fragments or their coalescence leads to compartmentalized stack formation without the participation of microtubules. These results demonstrate that this coalescence between isolated Golgi stacks is microtubule-independent and could thus be mediated by membranous tubules.

Effect of ion adsorption on the electrokinetic properties of erythrocytes.

The microelectrophoresis technique was used to determine the dependence of human erythrocyte surface potential on the concentration of various cations and anions. The interpretation of the results is based on the Gouy--Chapman--Stern theory. Values of pK, characterizing the binding of ions to the external surface of erythrocytes, as well as numbers of binding sites per unit area were determined. The affinities of ions for the red cell membrane were shown to decrease in the sequence: H+ greater than Ca2+ greater than Sr2+ greater than Mg2+ greater than Ba2+ greater than Li+ greater than Na+ congruent to congruent to K+ congruent to NH4+ and trinitrophenol greater than IO4- greater than CIO4- greater than salicylate congruent to I- greater than greater than SCN- greater than H2PO4- greater than Br- greater than Cl- greater than HPO4(2-). Changes in the ionic strength of the medium resulted in changes in numbers of exposed ion-binding sites. This phenomenon is interpreted in terms of ionic strength-dependent structural transformations of the cell surface coat.

[Structural changes in the contractile proteins of muscle fiber studied by polarization ultraviolet fluorescence microscopy. IX. The effect of the pH and ionic strength of the solution on the conformational restructurings of F-actin induced by the binding of heavy meromyosin]. / Izuchenie strukturnykh izmenenii sokratitel'nykh belkov myshechnogo volokna s pomoshch'iu poliarizatsionnoi ul'trafioletovoi fluorestsentnoi mikroskopii. IX. Vliianie pH i ionnoi sily rastvora na konformatsionnye perestroiki F-aktina, indutsirovannye sviazyvaniem tiazhelogo meromiozina.

The dependence of F-actin conformational changes induced by the F-actin-HMM complex on pH and ionic strength was found by polarized ultraviolet fluorescence microscopy. It is discovered that pH affects sufficiently the cooperativity of F-actin structural changes, while the ionic strength affects their depth. The actomyosin complex was supposed to be at least in two structural states, differing in their orientation as well as in flexibility of F-actin monomers.

The Cole-Moore effect in nodal membrane of the frog Rana ridibunda: evidence for fast and slow potassium channels.

The K conductance (gK) kinetics were studied in voltage-clamped frog nodes (Rana ridibunda) in double-pulse experiments. The Cole-Moore translation for gK--t curves associated with different initial potentials (E) was only observed with a small percentage of fibers. The absence of the translation was found to be caused by the involvement of an additional, slow, gK component. This component cannot be attributed to a multiple-state performance of the k channel. It can only be accounted for by a separate, slow K channel, the fast channel being the same as the n4 K channel in R. pipiens. The slow K channel is characterized by weaker sensitivity to TEA, smaller density, weaker potential (E) dependence, and somewhat more negative E range of activation than the fast K channel. According to characteristics of the slow K system, three types of fibers were found. In Type I fibers (most numerous) the slow K channel behaves as and n4 HH channel. In Type II fibers (the second largest group found) the slow K channel obeys the HH kinetics within a certain E range only; beyond this range the exponential decline of the slow gK component is preceded by an E-dependent delay, its kinetics after the delay being the same as those in Type I fibers. In Type III fibers (rare) the slow K channel is lacking, and it is only in these fibers that the Cole-Moore translation of the measured gK--t curves can be observed directly. The physiological role of the fast and slow K channel in amphibian nerves is briefly discussed.