Biomedical subjects

R S Polishchuk

Publications and source records attributed to R S Polishchuk.

At least 19 recordsLinked to original sources

PubMed2004

Structural aspects of Golgi function.

Since its discovery, the Golgi complex has attracted the attention of cell biologists because of its 'fashionable' morphology and central position within the secretory system of the cell. Here, we discuss how the three-dimensional architecture of the Golgi complex relates to its multiple functions in protein sorting and processing, and how an analysis of the morphology of the Golgi complex can help to provide an understanding of the mechanisms involved in transport through this unique organelle.

Animals↗

PubMed2001

Small cargo proteins and large aggregates can traverse the Golgi by a common mechanism without leaving the lumen of cisternae.

Procollagen (PC)-I aggregates transit through the Golgi complex without leaving the lumen of Golgi cisternae. Based on this evidence, we have proposed that PC-I is transported across the Golgi stacks by the cisternal maturation process. However, most secretory cargoes are small, freely diffusing proteins, thus raising the issue whether they move by a transport mechanism different than that used by PC-I. To address this question we have developed procedures to compare the transport of a small protein, the G protein of the vesicular stomatitis virus (VSVG), with that of the much larger PC-I aggregates in the same cell. Transport was followed using a combination of video and EM, providing high resolution in time and space. Our results reveal that PC-I aggregates and VSVG move synchronously through the Golgi at indistinguishable rapid rates. Additionally, not only PC-I aggregates (as confirmed by ultrarapid cryofixation), but also VSVG, can traverse the stack without leaving the cisternal lumen and without entering Golgi vesicles in functionally relevant amounts. Our findings indicate that a common mechanism independent of anterograde dissociative carriers is responsible for the traffic of small and large secretory cargo across the Golgi stack.

Animals↗

PubMed2001

Maintenance of Golgi structure and function depends on the integrity of ER export.

The Golgi apparatus comprises an enormous array of components that generate its unique architecture and function within cells. Here, we use quantitative fluorescence imaging techniques and ultrastructural analysis to address whether the Golgi apparatus is a steady-state or a stable organelle. We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model. Enzymes and recycling components are continuously exiting and reentering the Golgi apparatus by membrane trafficking pathways to and from the ER, whereas Golgi matrix proteins and coatomer undergo constant, rapid exchange between membrane and cytoplasm. When ER to Golgi transport is inhibited without disrupting COPII-dependent ER export machinery (by brefeldin A treatment or expression of Arf1[T31N]), the Golgi structure disassembles, leaving no residual Golgi membranes. Rather, all Golgi components redistribute into the ER, the cytoplasm, or to ER exit sites still active for recruitment of selective membrane-bound and peripherally associated cargos. A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region. In cells expressing Sar1[T39N], a constitutively inactive form of Sar1 that completely disrupts ER exit sites, Golgi glycosylation enzymes, matrix, and itinerant proteins all redistribute to the ER. These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis. Instead, they suggest that the Golgi complex is a dynamic, steady-state system, whose membranes can be nucleated and are maintained by the activities of the Sar1-COPII and Arf1-coatomer systems.

ADP-Ribosylation Factor 1↗

PubMed2001

Rapid cycling of lipid raft markers between the cell surface and Golgi complex.

The endocytic itineraries of lipid raft markers, such as glycosyl phosphatidylinositol (GPI)-anchored proteins and glycosphingolipids, are incompletely understood. Here we show that different GPI-anchored proteins have different intracellular distributions; some (such as the folate receptor) accumulate in transferrin-containing compartments, others (such as CD59 and GPI-linked green fluorescent protein [GFP]) accumulate in the Golgi apparatus. Selective photobleaching shows that the Golgi pool of both GPI-GFP and CD59-GFP constantly and rapidly exchanges with the pool of these proteins found on the plasma membrane (PM). We visualized intermediates carrying GPI-GFP from the Golgi apparatus to the PM and separate structures delivering GPI-GFP to the Golgi apparatus.GPI-GFP does not accumulate within endocytic compartments containing transferrin, although it is detected in intracellular structures which are endosomes by the criteria of accessibility to a fluid phase marker and to cholera and shiga toxin B subunits (CTxB and STxB, which are also found in rafts). GPI-GFP and a proportion of the total CTxB and STxB taken up into cells are endocytosed independently of clathrin-associated machinery and are delivered to the Golgi complex via indistinguishable mechanisms. Hence, they enter the Golgi complex in the same intermediates, get there independently of both clathrin and rab5 function, and are excluded from it at 20 degrees C and under conditions of cholesterol sequestration. The PM-Golgi cycling pathway followed by GPI-GFP could serve to regulate lipid raft distribution and function within cells.

Biological Transport↗

PubMed2000

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.

Animals↗

PubMed2000

Visualizing membrane traffic in vivo by combined video fluorescence and 3D electron microscopy.

In studies of dynamic cellular processes, it would be ideal to be able to combine the capability of in vivo fluorescence video microscopy with the power of resolution of electron microscopy (EM). This article describes an approach based on the association of these two techniques, by which an individual intracellular structure can be monitored in vivo, typically through the use of markers fused with green-fluorescent protein, and then analysed by EM and three-dimensional reconstruction methods, resulting in a 'snapshot' of its fine structure at any chosen time in its life cycle. The potential of this approach is discussed in relation to various aspects of cell biology and especially to the question of the morpho-functional organization of the intracellular membrane trafficking pathways.

Animals↗

PubMed1999

Golgi membranes are absorbed into and reemerge from the ER during mitosis.

Quantitative imaging and photobleaching were used to measure ER/Golgi recycling of GFP-tagged Golgi proteins in interphase cells and to monitor the dissolution and reformation of the Golgi during mitosis. In interphase, recycling occurred every 1.5 hr, and blocking ER egress trapped cycling Golgi enzymes in the ER with loss of Golgi structure. In mitosis, when ER export stops, Golgi proteins redistributed into the ER as shown by quantitative imaging in vivo and immuno-EM. Comparison of the mobilities of Golgi proteins and lipids ruled out the persistence of a separate mitotic Golgi vesicle population and supported the idea that all Golgi components are absorbed into the ER. Moreover, reassembly of the Golgi complex after mitosis failed to occur when ER export was blocked. These results demonstrate that in mitosis the Golgi disperses and reforms through the intermediary of the ER, exploiting constitutive recycling pathways. They thus define a novel paradigm for Golgi genesis and inheritance.

Animals↗

PubMed1999

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.

Actins↗

PubMed1995

Heterogeneity of smooth muscle cells in embryonic human aorta.

Cellular composition of aortas from 5- to 12-week and 18- to 28-week-old human embryos were investigated using immunocytochemistry, scanning and transmission electron microscopy. The aorta of the 5- to 12-week-old embryos consisted of three sublayers differing in cellular composition. The inner sublayer adjacent to the endothelium contained round and ovoid cells with synthetic phenotype. In the intermediate sublayer, spindle-like cells ultrastructurally similar to smooth muscle cells were found. Cells of the outer sublayer resembled fibroblasts or poorly differentiated mesenchymal cells. There were not definite morphological borders between sublayers. In the 18- to 28-week-old embryo aorta the intima was separated from media by internal elastic lamina. Intimal and innermost medial cells had predominately stellate shape and synthetic phenotype. The outer part of media contained spindle-like cells that had well developed contractile structures. Both the 5- to 12-week-old and the 18- to 28-week-old embryo aortic cells were positively stained for alpha-actin and myosin and negatively stained for macrophage antigens. Thus, the majority of embryo aortic cells appeared smooth muscle cells, however there was a regional difference in shape and synthetic state of these cells.

Aorta, Thoracic↗

PubMed1993

[Changes in the spatial organization and structure of the smooth-muscle cells of the tunica media of the major arteries during lengthwise stretching in situ].

It has been shown that in vivo stretching of the arterial wall leads to the loss of cell-cell contacts between medial smooth muscle cells (SMC), and to the change in the cell cooperation type. It is accompanied by the increase in SMC proliferative activity, and by changes in their shape and phenotype, from the contractile to the synthetic one. The increase in the number of extracellular connective tissue structures occurs in the intercellular space.

Animals↗

PubMed1993

[Cellular orientation and form in the elastic-hyperplastic layer of the human aortic intima in the area of the ostia of the intercostal arteries].

Using the Hatcher monolayer method, it has been shown that the aortic intimal smooth muscle cells, disposed on the flow divider of an intercostal artery, have predominantly spindle-like shapes to be oriented perpendicularly to the long axis of the vessel. Unlike, in the proximal and lateral sites of the intercostal ostium the intima is represented by stellate cells lacking any orientation. These distinctions may be presumably determined by the irregular distribution of hemodynamic loading on the aortic intima in different regions around the ostium of an intercostal artery.

Adult↗

PubMed1993

[Changes in the orientation of the smooth-muscle cells of the tunica media from major arteries under constant lengthwise stretching in situ].

It has been shown that changes in the orientation of arterial smooth muscle cells during a constant longitudinal stretching of the artery in vivo are not similar in different sections of the stretching zone. Cells in the proximal and distal sections keep their orientation but this orientation differs from that of smooth muscle cells in the control arteries. Cells in the central part of the stretching region lose their definite orientation to settle randomly.

Animals↗

PubMed1991